Analysis of antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery.

PURPOSE: To analyze antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery with the focus on coagulase-negative staphylococci (CNS) during cataract surgery and discuss the implications of this colonization as a potential risk of acquiring endophthalmitis. METHODS: After approval of the institutional review board and informed consent from patients had been obtained, conjunctival swabs for culture from 59 patients undergoing cataract surgery were taken of the fellow eye at baseline (C0) and from the eye to be operated before (T0) and after (T1) irrigation with povine-iodine 5%, and at the end of surgery (T2). Genes responsible for virulence (mecA, ica and atlE) and antibiotic profile were determined; strain clonality of persistent colonizing Staphylococcus epidermidis strains was established by the Multi-locus sequence typing (MLST). RESULTS: The frequency of CNS was significantly reduced in T1 (13.6%) from 81.4% in T0 and 86.4% in C0. The frequency of mecA, ica and atlE genes was 34.4%, 37.5% and 61.4%, respectively; and methicillin phenotypic resistance was 35.4%. S. epidermidis was the most frequent species isolated in every time point. MLST revealed in 7 patients 100% coincidence of the seven alleles of the S. epidermidis isolated previous to povine-iodine 5% disinfection and at the end of the surgery. CNS isolates from T1 or T2 corresponded to the same species, antibiotic and virulence profile as those isolates from C0 or T0. CONCLUSION: Povidone-iodine 5% prophylaxis before surgery significantly reduced conjunctival contamination; in those that persisted, the source of contamination was mostly the patient's microbiota confirmed by the MLST system.

Restricted X chromosome introgression and support for Haldane's rule in hybridizing damselflies.

Contemporary hybrid zones act as natural laboratories for the investigation of species boundaries and may shed light on the little understood roles of sex chromosomes in species divergence. Sex chromosomes are considered to function as a hotspot of genetic divergence between species; indicated by less genomic introgression compared to autosomes during hybridization. Moreover, they are thought to contribute to Haldane's rule, which states that hybrids of the heterogametic sex are more likely to be inviable or sterile. To test these hypotheses, we used contemporary hybrid zones of Ischnura elegans, a damselfly species that has been expanding its range into the northern and western regions of Spain, leading to chronic hybridization with its sister species Ischnura graellsii. We analysed genome-wide SNPs in the Spanish I. elegans and I. graellsii hybrid zone and found (i) that the X chromosome shows less genomic introgression compared to autosomes, and (ii) that males are underrepresented among admixed individuals, as predicted by Haldane's rule. This is the first study in Odonata that suggests a role of the X chromosome in reproductive isolation. Moreover, our data add to the few studies on species with X0 sex determination system and contradict the hypothesis that the absence of a Y chromosome causes exceptions to Haldane's rule.

Genome assembly, sex-biased gene expression and dosage compensation in the damselfly Ischnura elegans.

The evolution of sex chromosomes, and patterns of sex-biased gene expression and dosage compensation, are poorly known among early winged insects such as odonates. We assembled and annotated the genome of Ischnura elegans (blue-tailed damselfly), which, like other odonates, has a male-hemigametic sex-determining system (X0 males, XX females). By identifying X-linked genes in I. elegans and their orthologs in other insect genomes, we found homologies between the X chromosome in odonates and chromosomes of other orders, including the X chromosome in Coleoptera. Next, we showed balanced expression of X-linked genes between sexes in adult I. elegans, i.e. evidence of dosage compensation. Finally, among the genes in the sex-determining pathway only fruitless was found to be X-linked, while only doublesex showed sex-biased expression. This study reveals partly conserved sex chromosome synteny and independent evolution of dosage compensation among insect orders separated by several hundred million years of evolutionary history.

Next-generation sequencing, isolation and characterization of 14 microsatellite loci of Canthon cyanellus (Coleoptera: Scarabaeidae).

BACKGROUND: We used Illumina paired-end sequencing to isolate and characterize microsatellites of Canthon cyanellus, a Neotropical roller dung beetle, encompassing several lineages within its distribution range. METHODS AND RESULTS: We examined C. cyanellus specimens collected at eight different localities in Mexico (two or three specimens per locality). We initially performed amplification tests with 16 loci, but two of which were unsuccessful. The 14 remaining microsatellites were polymorphic, with 2-16 alleles each. The expected and observed heterozygosity ranged from 0.11 to 0.76 and from 0.20 to 0.78, respectively. CONCLUSIONS: These microsatellites will help to assess structure at the population and lineage levels, identify zones of potential hybridization between lineages, and draw a more precise geographic delimitation of C. cyanellus lineages.

Serotypes of Streptococcus pneumoniae in Children Aged <5 Years Hospitalized With or Without Pneumonia in Developing and Emerging Countries: A Descriptive, Multicenter Study.

BACKGROUND: Improving knowledge regarding Streptococcus pneumoniae distribution in pneumonia cases is important to better target preventive and curative measures. The objective was to describe S. pneumoniae serotypes in children with or without pneumonia. METHODS: It was a case-control study carried out in 8 developing and emerging countries between 2010 and 2014. Cases were children aged <5 years admitted to the hospital for pneumonia. Controls were children admitted for surgery or routine outpatient care. RESULTS: In nasopharyngeal samples, S. pneumoniae were detected in 68.2% of the cases and 47.5% of the controls (P < .001). Nasopharyngeal carriage was associated with a higher risk of being a case in 6/8 study sites (adjusted odds ratio ranged from 0.71 [95% confidence interval [CI], .39-1.29; P = .26] in India [Pune/Vadu] to 11.86 [95% CI, 5.77-24.41; P < .001] in Mongolia). The 13-valent pneumococcal conjugate vaccine (PCV13) serotypes were more frequently detected in cases with nasopharyngeal carriage (67.1%) than in controls with nasopharyngeal carriage (54.6%), P < .001. Streptococcus pneumoniae was detected in blood by polymerase chain reaction in 8.3% of the cases. Of 34 cases with an S. pneumoniae serotype detected in blood, 27 (79%) had the same serotype in the nasopharyngeal sample. CONCLUSIONS: The results confirm the assumption that the isolate carrying or causing disease in an individual is of the same serotype. Most serotypes independently associated with nasopharyngeal carriage or pneumonia are covered by PCV13, suggesting that increased PCV coverage would reduce the burden of S. pneumoniae-related pneumonia.

PRDM9 Diversity at Fine Geographical Scale Reveals Contrasting Evolutionary Patterns and Functional Constraints in Natural Populations of House Mice.

One of the major challenges in evolutionary biology is the identification of the genetic basis of postzygotic reproductive isolation. Given its pivotal role in this process, here we explore the drivers that may account for the evolutionary dynamics of the PRDM9 gene between continental and island systems of chromosomal variation in house mice. Using a data set of nearly 400 wild-caught mice of Robertsonian systems, we identify the extent of PRDM9 diversity in natural house mouse populations, determine the phylogeography of PRDM9 at a local and global scale based on a new measure of pairwise genetic divergence, and analyze selective constraints. We find 57 newly described PRDM9 variants, this diversity being especially high on Madeira Island, a result that is contrary to the expectations of reduced variation for island populations. Our analysis suggest that the PRDM9 allelic variability observed in Madeira mice might be influenced by the presence of distinct chromosomal fusions resulting from a complex pattern of introgression or multiple colonization events onto the island. Importantly, we detect a significant reduction in the proportion of PRDM9 heterozygotes in Robertsonian mice, which showed a high degree of similarity in the amino acids responsible for protein-DNA binding. Our results suggest that despite the rapid evolution of PRDM9 and the variability detected in natural populations, functional constraints could facilitate the accumulation of allelic combinations that maintain recombination hotspot symmetry. We anticipate that our study will provide the basis for examining the role of different PRDM9 genetic backgrounds in reproductive isolation in natural populations.

The evolutionary history of colour polymorphism in Ischnura damselflies.

A major challenge in evolutionary biology consists of understanding how genetic and phenotypic variation is created and maintained. In this study, we investigated the origin(s) and evolutionary patterns of the female-limited colour polymorphism in ischnuran damselflies. These consist of the presence of one to three colour morphs: one androchrome morph with a coloration that is similar to the male and two gynochrome morphs (infuscans and aurantiaca) with female-specific coloration. We (i) documented the colour and mating system of 44 of the 75 taxa within the genus Ischnura, (ii) reconstructed the evolutionary history of colour and mating system to identify the ancestral state, (iii) evaluated the stability of the colour morph status over time and (iv) tested for a correlation between colour and mating system. We found that the ancestral female colour of Ischnura was monomorphic and aurantiaca and that colour morph status changed over time, characterized by many gains and losses across the species tree. Our results further showed that colour polymorphism is significantly more frequent among polyandric species, whereas monandric species tend to be monomorphic. Research on some Ischnura species has shown that colour morphs have evolved to reduce male mating harassment, and our finding that the same phenotypic morphs have evolved multiple times (convergent evolution) suggests that several species in this genus might be experiencing similar selective pressures.

[Lithogenic risk in patients from Paraguay with urolithiasis]. / Perfil de riesgo litogénico en pacientes con urolitiasis en Paraguay.

UNLABELLED: The lithogenic risk profile is a graphical representation of metabolic factors and urinary saturation involved in the stone formation with their respective critical values. AIM: To determine the lithogenic risk profile in patients with urolithiasis. MATERIAL AND METHODS: Personal data such as anthropometric, history of diseases and family history of urolithiasis were recorded. Different compounds acting as promoters or inhibitors of crystallization were measured in serum and urine samples, and the data obtained were used to calculate urinary saturation using Equil software. RESULTS: We included 30 men and 43 women with a median age of 45 (34-54) years. Overweight and family history of urolithiasis was reported in 63 and 32% respectively. Crystallization risk was detected in 74% of participants. The most common urinary abnormalities were hypocitraturia in 48% and hypercalciuria in 40%. CONCLUSIONS: The lithogenic profile revealed urinary saturation compatible with crystallization risk in 74% of the studied patients.

Plasticity and associated epigenetic mechanisms play a role in thermal evolution during range expansion.

Due to global change, many species are shifting their distribution and are thereby confronted with novel thermal conditions at the moving range edges. Especially during the initial phases of exposure to a new environment, it has been hypothesized that plasticity and associated epigenetic mechanisms enable species to cope with environmental change. We tested this idea by capitalizing on the well-documented southward range expansion of the damselfly Ischnura elegans from France into Spain where the species invaded warmer regions in the 1950s in eastern Spain (old edge region) and in the 2010s in central Spain (new edge region). Using a common garden experiment at rearing temperatures matching the ancestral and invaded thermal regimes, we tested for evolutionary changes in (thermal plasticity in) larval life history and heat tolerance in these expansion zones. Through the use of de- and hypermethylating agents, we tested whether epigenetic mechanisms play a role in enabling heat tolerance during expansion. We used the phenotype of the native sister species in Spain, I. graellsii, as proxy for the locally adapted phenotype. New edge populations converged toward the phenotype of the native species through plastic thermal responses in life history and heat tolerance while old edge populations (partly) constitutively evolved a faster life history and higher heat tolerance than the core populations, thereby matching the native species. Only the heat tolerance of new edge populations increased significantly when exposed to the hypermethylating agent. This suggests that the DNA methylation machinery is more amenable to perturbation at the new edge and shows it is able to play a role in achieving a higher heat tolerance. Our results show that both (evolved) plasticity as well as associated epigenetic mechanisms are initially important when facing new thermal regimes but that their importance diminishes with time.

Genomic epidemiology of the primary methicillin-resistant Staphylococcus aureus clones causing invasive infections in Paraguayan children.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major human pathogens. It could carry numerous resistance genes and virulence factors in its genome, some of which are related to the severity of the infection. An observational, descriptive, cross-sectional study was designed to molecularly analyze MRSA isolates that cause invasive infections in Paraguayan children from 2009 to 2013. Ten representative MRSA isolates of the main clonal complex identified were analyzed with short-read paired-end sequencing and assessed for the virulome, resistome, and phylogenetic relationships. All the genetically linked MRSA isolates were recovered from diverse clinical sources, patients, and hospitals at broad gap periods. The pan-genomic analysis of these clones revealed three major and different clonal complexes (CC30, CC5, and CC8), each composed of clones closely related to each other. The CC30 genomes prove to be a successful clone, strongly installed and disseminated throughout our country, and closely related to other CC30 public genomes from the region and the world. The CC5 shows the highest genetic variability, and the CC8 carried the complete arginine catabolic mobile element (ACME), closely related to the USA300-NAE-ACME+, identified as the major cause of CA-MRSA infections in North America. Multiple virulence and resistance genes were identified for the first time in this study, highlighting the complex virulence profiles of MRSA circulating in the country. This study opens a wide range of new possibilities for future projects and trials to improve the existing knowledge on the epidemiology of MRSA circulating in Paraguay. IMPORTANCE: The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide. The most frequent MRSA clones identified in Paraguay in previous studies (including community and hospital acquired) were the Pediatric (CC5-ST5-IV), the Cordobes-Chilean (CC5-ST5-I), the SouthWest Pacific (CC30-ST30-IV), and the Brazilian (CC8-ST239-III) clones. In this study, the pan-genomic analysis of the most representative MRSA clones circulating in invasive infection in Paraguayan children over the years 2009-2013, such as the CC30-ST30-IV, CC5-ST5-IV, and CC8-ST8-IV, was carried out to evaluate their genetic diversity, their repertoire of virulence factors, and antimicrobial resistance determinants. This revealed multiple virulence and resistance genes, highlighting the complex virulence profiles of MRSA circulating in Paraguay. Our work is the first genomic study of MRSA in Paraguay and will contribute to the development of genomic surveillance in the region and our understanding of the global epidemiology of this pathogen.

The Role of Hybridization in Species Formation and Persistence.

Hybridization, or interbreeding between different taxa, was traditionally considered to be rare and to have a largely detrimental impact on biodiversity, sometimes leading to the breakdown of reproductive isolation and even to the reversal of speciation. However, modern genomic and analytical methods have shown that hybridization is common in some of the most diverse clades across the tree of life, sometimes leading to rapid increase of phenotypic variability, to introgression of adaptive alleles, to the formation of hybrid species, and even to entire species radiations. In this review, we identify consensus among diverse research programs to show how the field has progressed. Hybridization is a multifaceted evolutionary process that can strongly influence species formation and facilitate adaptation and persistence of species in a rapidly changing world. Progress on testing this hypothesis will require cooperation among different subdisciplines.

Ontogenetic shifts in male mating preference and morph-specific polyandry in a female colour polymorphic insect.

BACKGROUND: Sexual conflict over mating rates may favour the origin and maintenance of phenotypes with contrasting reproductive strategies. The damselfly Ischnura elegans is characterised by a female colour polymorphism that consists of one androchrome and two gynochrome female morphs. Previous studies have shown that the polymorphism is genetic and to a high extent maintained by negative frequency-dependent mating success that varies temporally and spatially. However, the role of learning in male mating preferences has received little attention. We used molecular markers to investigate differences in polyandry between female morphs. In addition, we experimentally investigated innate male mating preferences and experience-dependent shifts in male mating preferences for female morphs. RESULTS: Field and molecular data show that androchrome females were less polyandrous than gynochrome females. Interestingly, we found that naïve males showed significantly higher sexual preferences to androchrome than to gynochrome females in experimental trials. In contrast, experienced males showed no preference for androchrome females. CONCLUSIONS: The ontogenetic change in male mate preferences occurs most likely because of learned mate recognition after experience with females, which in this case does not result in a preference for one of the morphs, but rather in the loss of an innate preference for androchrome females.

[Clinically significant coagulase-negative staphylococci: most frequent species and virulence factors]. / Staphylococcus coagulasa-negativa clínicamente significativos: Especies más frecuentes y factores de virulencia.

BACKGROUND: Coagulase-negative staphylococci have emerged as responsible for a large number of infections. However, it is often difficult to assess its pathogenic role or to discard it as a contaminant. AIM: The goal of this study was to identify clinically significant coagulase-negative staphylococci to the species level and their virulence factors. Isolates came from patients consulting at the San Roque Laboratory from 2009 to 2011. MATERIAL AND METHODS: Species identification was performed by De Paulis et al simplified method. Production of biofilm, hemolysins, lipases, lecithinases and DNase were determined by conventional methods; methicillin-resistance by diffusion method and mecA and Panton-Valentine genes, by multiplex PCR. RESULTS: Out of 64 isolates, 40.6% were S. epidermidis; 20.3%, S. haemolyticus, and 15.6%, S. lugdunensis. Biofilm production was detected in 73.1% of S. epidermidis, 53.8% of S. haemolyticus and 40% of S. lugdunensis. mecA gene was identified in 69.2% of S. epidermidis, 92.3% of S. haemolyticus and none of S. lugdunensis. 83% of mecA (+) S. epidermidis isolates were biofilm producers as compared to 50% of the mecA (-). CONCLUSION: The frequency of S. lugdunensis, the most virulent coagulase-negative staphylococci species, was relatively high. The main virulence factor in S. epidermidis was biofilm production, being higher in those resistant to methicillin.

Genomic epidemiology of Staphylococcus aureus isolated from bloodstream infections in South America during 2019 supports regional surveillance.

Staphylococcus aureus remains one of the leading causes of infections worldwide and a common cause of bacteraemia. However, studies documenting the epidemiology of S. aureus in South America using genomics are scarce. We hereby report on the largest genomic epidemiology study to date of both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in South America, conducted by the StaphNET-SA network. We characterised 404 genomes recovered from a prospective observational study of S. aureus bacteraemia in 58 hospitals from Argentina, Bolivia, Brazil, Paraguay and Uruguay between April and October 2019. We show that a minority of S. aureus isolates are phenotypically multi-drug resistant (5.2%), but more than a quarter are resistant to macrolide-lincosamide-streptogramin B (MLSb). MSSA were more genetically diverse than MRSA. Lower rates of associated antimicrobial resistance in community-associated(CA)-MRSA versus hospital-associated (HA)-MRSA were found in association with three S. aureus genotypes dominating the MRSA population: CC30-MRSA-IVc-t019-lukS/F-PV+, CC5-MRSA-IV-t002-lukS/F-PV- and CC8-MRSA-IVc-t008-lukS/F-PV+-COMER+. These are historically from a CA origin, carry on average fewer antimicrobial resistance determinants, and often lack key virulence genes. Surprisingly, CC398-MSSA-t1451-lukS/F-PV- related to the CC398 human-associated lineage is widely disseminated throughout the region, and is described here for the first time as the most prevalent MSSA lineage in South America. Moreover, CC398 strains carrying ermT (largely responsible for the MLSb resistance rates of MSSA strains: inducible iMLSb phenotype) and sh_fabI (related to triclosan resistance) were recovered from both CA and HA origin. The frequency of MRSA and MSSA lineages differed between countries but the most prevalent S. aureus genotypes are high-risk clones widely distributed in the South American region without a clear country-specific phylogeographical structure. Therefore, our findings underline the need for continuous genomic surveillance by regional networks such as StaphNET-SA. This article contains data hosted by Microreact.

Terpenes Combinations Inhibit Biofilm Formation in Staphyloccocus aureus by Interfering with Initial Adhesion.

The biofilm is a conglomerate of cells surrounded by an extracellular matrix, which contributes to the persistence of infections. The difficulty in removing the biofilm drives the research for new therapeutic options. In this work, the effect of terpenes (−)-trans-Caryophyllene, (S)-cis-Verbenol, (S)-(−)-Limonene, (R)-(+)-Limonene, and Linalool was evaluated, individually and in combinations on bacterial growth, by assay with resazurin; the formation of biofilm, by assay with violet crystal; and the expression of associated genes, by real-time PCR, in two clinical isolates of Staphyloccocus aureus, ST30-t019 and ST5-t311, responsible for more than 90% of pediatric infections by this pathogen in Paraguay. All combinations of terpenes can inhibit biofilm formation in more than 50% without affecting bacterial growth. The most effective combination was (−)-trans-Caryophyllene and Linalool at a 500 µg/mL concentration for each, with an inhibition percentage of 88%. This combination decreased the expression levels of the sdrD, spa, agr, and hld genes associated with the initial cell adhesion stage and quorum sensing. At the same time, it increased the expression levels of the cap5B and cap5C genes related to the production of capsular polysaccharides. The combinations of compounds tested are promising alternatives to inhibit biofilm formation in S. aureus.

In vitro antibacterial activity, biofilm formation inhibition and chromatographic profile of methanolic extracts of two Pterocaulon species against MRSA.

The methanolic extracts of Pterocaulon alopecuroides and Pterocaulon angustifolium were assayed for antibacterial activity and biofilm formation inhibition of four community-acquired-MRSA isolates representative of ST30 t975, ST30 t021, ST5 t311, and ST4335 t008 clones that are responsible for invasive infections in Paraguayan children. Both Pterocaulon extracts showed significant antibacterial activity with a minimum inhibitory concentration of 200 µg/mL against the four isolates. P. angustifolium showed inhibition of biofilm formation for the four isolates, whereas P. alopecuroides showed inhibition for three of them. The chemical constituents were identified by liquid chromatography coupled to tandem mass spectrometry. Phenolic compounds were detected in the two species as well as coumarins. These results showed that these plants are sources of compounds with activity against MRSA.

The evolutionary outcomes of climate-change-induced hybridization in insect populations.

Rapid range shifts are one of the most frequent responses to climate change in insect populations. Climate-induced range shifts can lead to the breakdown of isolation barriers, and thus, to an increase in hybridization and introgression. Long-term evolutionary consequences such as the formation of hybrid zones, introgression, speciation, and extinction have been predicted as a result of climate-induced hybridization. Our review shows that there has been an increase in the number of published cases of climate-induced hybridization in insects, and that the formation of hybrid zones and introgression seems to be, at the moment, the most frequent outcomes. Although introgression is considered positive, since it increases species' genetic diversity, in the long term, it could lead to negative outcomes such as species fusion or genetic swamping.

Convergence of life history and physiology during range expansion toward the phenotype of the native sister species.

In our globally changing planet many species show range expansions whereby they encounter new thermal regimes that deviate from those of their source region. Pressing questions are to what extent and through which mechanisms, plasticity and/or evolution, species respond to the new thermal regimes and whether these trait changes are adaptive. Using a common-garden experiment, we tested for plastic and evolutionary trait changes in life history and a set of understudied biochemical/physiological traits during the range expansion of the damselfly Ischnura elegans from France into a warmer region in Spain. To assess the adaptiveness of the trait changes we used the phenotype of its native sister species in Spain, I. graellsii, as proxy for the locally adapted phenotype. While our design cannot fully exclude maternal effects, our results suggest that edge populations adapted to the local conditions in the newly invaded region through the evolution of a faster pace-of-life (faster development and growth rates), a smaller body size, a higher energy budget and increased expression levels of the heat shock gene DnaJ. Notably, based on convergence toward the phenotype of the native sister species and its thermal responses, and the fit with predictions of life history theory these potential evolutionary changes were likely adaptive. Nevertheless, the convergence toward the native sister species is incomplete for thermal plasticity in traits associated with anaerobic metabolism and melanization. Our results highlight that evolution might at least partly contribute in an adaptive way to the persistence of populations during range expansion into new thermal environments and should be incorporated when predicting and understanding species' range expansions.

Nasopharyngeal Viral and Bacterial Co-Detection among Children from Low- and Middle-Income Countries with and without Pneumonia.

The role of microbial coinfection in the pathogenesis of pneumonia in children is not well known. The aim of this work was to describe the prevalence of microorganism co-detection in nasopharyngeal samples (NPS) of pneumonia cases and control subjects and to study the potential association between nasopharyngeal microorganism co-detection and pneumonia. A case-control study was carried out from 2010 to 2014 in nine study sites located in low- or middle-income countries. The data from 888 children under 5 years of age with pneumonia (cases) and 870 children under 5 without pneumonia (controls) were analyzed. Nasopharyngeal samples were collected; reverse transcription polymerase chain reaction (RT-PCR) enabled the detection of five bacteria and 19 viruses. Multiple, mixed-effects logistic regression modeling was undertaken to evaluate the association between microorganism co-detection and pneumonia. A single Streptococcus pneumoniae colonization was observed in 15.2% of the controls and 10.1% of the cases (P = 0.001), whereas S. pneumoniae and a single virus co-detection was observed in 33.3% of the cases and in 14.6% of the controls (P < 0.001). Co-detections with rhinovirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, and influenza virus were more frequent in the cases compared with the controls (P < 0.001) and were significantly associated with pneumonia in multiple regression analysis. The proportion of single virus detection without bacterial co-detection was not different between cases and controls (13.6% versus 11.3%, P = 0.13). This study suggests that coinfection of S. pneumoniae and certain viruses may play a role in the pathophysiology of pneumonia in children.

Ure2 is involved in nitrogen catabolite repression and salt tolerance via Ca2+ homeostasis and calcineurin activation in the yeast Hansenula polymorpha.

Disruption of HpURE2 resulted in a low expression of genes encoding nitrate-assimilatory proteins; sensitivity to Li(+), Na(+), and Cd(2+); no induction of ENA1; low levels of the GATA-type transcription factor Gat1; and low intracellular Ca(2+) levels. Gat1 levels were also very low in a Δcnb1 mutant lacking the regulatory subunit of calcineurin. The strain Δure2 was very sensitive to the calcineurin inhibitor FK506 and displayed several phenotypes reminiscent of Δcnb1. The reporter 4xCDRE-lacZ, containing calcineurin-dependent response elements in its promoter, revealed that calcineurin activation was reduced in HpΔure2. Expression of ScURE2 in Δure2 rescued nitrogen catabolite repression and Cd(2+) tolerance but not those phenotypes depending on calcineurin activation, such as salt tolerance and nitrate assimilation gene derepression. HpΔure2 showed an increased expression of the gene PMR1 encoding the Golgi Ca(2+)-ATPase, whereas that of PMC1 encoding the vacuolar Ca(2+)-ATPase remained unaltered. PMR1 up-regulation was abolished by deletion of the GATA-type transcription factor GAT2 in a HpΔure2 genetic background, and normal Ca(2+) levels were recovered. Moreover, overexpression of GAT2 or PMR1 yielded strains mimicking the phenotype of the HpΔure2. This suggests that the low Ca(2+) levels in the HpΔure2 mutant are due to the high levels of Pmr1 that replenish the Golgi Ca(2+) content, thus acting as a negative signal for Ca(2+) entry into the cell. We conclude that HpUre2 is involved in salt tolerance and also in nitrate assimilation gene derepression via Ca(2+) homeostasis regulation and calcineurin activation, which control the levels of Gat1.