Harmonisation of in-silico next-generation sequencing based methods for diagnostics and surveillance.

Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype-phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.

Substance use patterns in 9-10 year olds: Baseline findings from the adolescent brain cognitive development (ABCD) study.

BACKGROUND: The Adolescent Brain Cognitive Development ™ Study (ABCD Study®) is an open-science, multi-site, prospective, longitudinal study following over 11,800 9- and 10-year-old youth into early adulthood. The ABCD Study aims to prospectively examine the impact of substance use (SU) on neurocognitive and health outcomes. Although SU initiation typically occurs during teen years, relatively little is known about patterns of SU in children younger than 12. METHODS: This study aims to report the detailed ABCD Study® SU patterns at baseline (n = 11,875) in order to inform the greater scientific community about cohort's early SU. Along with a detailed description of SU, we ran mixed effects regression models to examine the association between early caffeine and alcohol sipping with demographic factors, externalizing symptoms and parental history of alcohol and substance use disorders (AUD/SUD). PRIMARY RESULTS: At baseline, the majority of youth had used caffeine (67.6 %) and 22.5 % reported sipping alcohol (22.5 %). There was little to no reported use of other drug categories (0.2 % full alcohol drink, 0.7 % used nicotine, <0.1 % used any other drug of abuse). Analyses revealed that total caffeine use and early alcohol sipping were associated with demographic variables (p's<.05), externalizing symptoms (caffeine p = 0002; sipping p = .0003), and parental history of AUD (sipping p = .03). CONCLUSIONS: ABCD Study participants aged 9-10 years old reported caffeine use and alcohol sipping experimentation, but very rare other SU. Variables linked with early childhood alcohol sipping and caffeine use should be examined as contributing factors in future longitudinal analyses examining escalating trajectories of SU in the ABCD Study cohort.

Complete genome sequence of the animal pathogen Listeria ivanovii, which provides insights into host specificities and evolution of the genus Listeria.

We report the complete and annotated genome sequence of the animal pathogen Listeria ivanovii subsp. ivanovii strain PAM 55 (serotype 5), isolated in 1997 in Spain from an outbreak of abortion in sheep. The sequence and its analysis are available at an interactive genome browser at the Institut Pasteur (http://genolist.pasteur.fr/LivaList/).

Involvement of closely related strains of a new clonal group of Listeria monocytogenes in the 1998-99 and 2002 multistate outbreaks of foodborne listeriosis in the United States.

In 1998-99, a multistate outbreak of listeriosis in the United States was associated with contaminated hot dogs and was caused by a strain of Listeria monocytogenes serotype 4b that had been only rarely encountered before in the national PulseNet database. Upon further characterization, the strains from this outbreak were designated as Epidemic Clone II (ECII). ECII isolates exhibited diversification in a genomic region ("region 18") that was otherwise conserved among L. monocytogenes of serotype 4b. Additional unique genetic markers were identified through genome sequencing of one of the isolates from the 1998-99 outbreak. In 2002, another multistate outbreak of listeriosis also involved bacteria of serotype 4b and was attributed to contaminated turkey deli meats. Molecular subtyping data revealed that the macrorestriction patterns of the isolates from the 1998-99 and 2002 outbreaks were closely related. In addition, the 2002 outbreak isolates harbored chromosomal genetic markers found to be unique to, and typical of, the 1998-99 outbreak isolates, including diversification in genomic region 18. Macroarray- based subtyping using chromosomal sequences confirmed the close genetic relatedness between the isolates from the two outbreaks. Genomic content was highly conserved among isolates from each outbreak, with differences detected only in prophage and internalin-like gene sequences. However, since these differences were observed among isolates from each of the outbreaks, they did not differentiate the 1998-99 isolates as a group from those of the 2002 outbreak. Two of 15 randomly chosen serotype 4b clinical isolates from a non-outbreak period (calendar year 2003) appeared to be closely related to the 1998-99 and 2002 outbreak isolates. These findings suggest that both multistate outbreaks of listeriosis in the United States involved closely related members of a single clonal group (ECII) that had not been identified in outbreaks prior to 1998. Since the outbreaks involved different food vehicles and processing plants, the findings suggest establishment of ECII in a still unidentified reservoir in the United States, from which the organisms were introduced to different processing plants.

Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola.

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.

Assessment of the rind microbial diversity in a farmhouse-produced vs a pasteurized industrially produced soft red-smear cheese using both cultivation and rDNA-based methods.

AIMS: The diversity of the surface flora of two French red-smear soft cheeses was examined by cultivation-dependent and cultivation-independent methods to assess their composition and to evaluate the accuracy of both approaches. METHODS AND RESULTS: Culture-independent methods used involved 16S ribosomal DNA gene cloning and sequencing and single-strand conformation polymorphism analysis (SSCP). The culture-dependent method used involved direct culture and macroscopic observation, polymerase chain reaction of the 16S rRNA gene from DNA extracted from single colonies followed by complete sequencing of the gene. Only few species were recovered by both approaches either in the pasteurized and the farmer cheese. A large diversity of isolates or 16S rDNA sequences related to marine bacteria was identified at the surface of both cheeses. CONCLUSIONS: The results indicated that all three techniques were informative and complementary to allow a more accurate representativeness of the cheese surface biodiversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Cultivation and molecular methods have to be combined in order to obtain an extended view of the bacterial populations of complex ecosystems.

CAAT-Box, Contigs-Assembly and Annotation Tool-Box for genome sequencing projects.

MOTIVATION: Contigs-Assembly and Annotation Tool-Box (CAAT-Box) is a software package developed for the computational part of a genome project where the sequence is obtained by a shotgun strategy. CAAT-Box contains new tools to predict links between contigs by using similarity searches with other whole genome sequences. Most importantly, it allows annotation of a genome to commence during the finishing phase using a gene-oriented strategy. For this purpose, CAAT-Box creates an Individual Protein file (IPF) for each ORF of an assembly. The nucleotide sequence reported in an IPF corresponds to the sequence of the ORF with 500 additional bases before the ORF and 200 bases after. For annotation, additional information like Blast results can be added or linked to the IPFs as well as automatic and/or manual annotations. When a new assembly is performed, CAAT-Box creates new IPFs according to the old IPF panel. CAAT-Box recognizes the modified IPFs which are the only ones used for a new automatic analysis after each assembly. Using this strategy, the user works with a group of IPFs independently of the closure phase progression. The IPFs are accessible by a web server and can therefore be modified and commented by different groups. RESULT: CAAT-Box was used to obtain and to annotate several complete genomes like Listeria monocytogenes or Streptococcus agalactiae. AVAILABILITY: The program may be obtained from the authors and is freely available to non-profit organisations.

Modulation of anaerobic energy metabolism of Bacillus subtilis by arfM (ywiD).

Bacillus subtilis grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes. The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation. Mutation of the open reading frame ywiD located upstream of the respiratory nitrate reductase operon narGHJI resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth. Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged. Anaerobic induction of nasDE expression was found to be significantly dependent on intact ywiD, while anaerobic narGHJI expression was ywiD independent. Anaerobic transcription of hmp, encoding a flavohemoglobin-like protein, and of the fermentative operons lctEP and alsSD, responsible for lactate and acetoin formation, was partially dependent on ywiD. Expression of pta, encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced by ywiD. Transcription of the ywiD gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site. Anaerobic induction of ywiD by resDE was found to be indirect via resDE-dependent activation of fnr. The ywiD gene is subject to autorepression and nitrite repression. These results suggest a ResDE --> Fnr --> YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of B. subtilis. Therefore, ywiD was renamed arfM for anaerobic respiration and fermentation modulator.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Characterization of glucose-repression-resistant mutants of Bacillus subtilis: identification of the glcR gene.

In Bacillus subtilis, carbon catabolite repression (CCR) is mediated by the pleiotropic repressor CcpA and by ATP-dependent phosphorylation of the HPr protein of the phosphotransferase system (PTS). In this study, we attempted to identify novel genes that are involved in the signal transduction pathway that ultimately results in CCR in the presence of repressing carbon sources such as glucose. Seven mutants resistant to glucose repression of the levanase operon were isolated and characterized. All mutations were trans-acting and pleiotropic as determined by analyzing CCR of beta-xylosidase and of the sacPA and bglPH operon. Moreover, all mutations specifically affected repression exerted by glucose but not by other sugars. The mutations were mapped to three different loci on the genetic map, ptsG, glcR, and pgi. These three genes encode proteins involved in glucose metabolism. A novel repressor gene, glcR (ywpI), defined by two mutations, was studied in more detail. The glcR mutants exhibit loss of glucose repression of catabolic operons, a deficiency in glucose transport, and absence of expression of the ptsG gene. The mutant GlcR proteins act as super-repressors of ptsG expression.

Genetic and physical delineation of the region overlapping the progressive motor neuropathy (pmn) locus on mouse chromosome 13.

The mouse autosomal recessive mutation progressive motor neuropathy (pmn) results in early onset motor neuron disease with rapidly progressing hindlimb paralysis, severe muscular wasting, and death at 4--6 weeks of age. pmn is thus considered a good animal model for motor neuron diseases and the characterization of the causative gene should help in understanding the biological causes of human spinal muscular atrophies. Here we report the generation of a physical map based on a high-resolution and high-density genetic map encompassing the pmn locus on mouse chromosome 13. We have positioned the pmn locus and a cluster of markers cosegregating with it within a genetic interval of 0.30 cM, delineated by two clusters of markers. We have constructed an approximately 850-kb contig of BACs spanning the pmn critical region. This BAC contig contains the breakpoint of synteny between mouse chromosome 13 and human 1q and 7p regions and lays the foundation for identifying at the molecular level such a breakpoint region. The physical and genetic maps provided a support for the identification of five transcription units positioned in the nonrecombinant interval, and constitute invaluable tools for the identification of other candidate genes for the pmn mutation.

Gene expression profiles in normal and Otx2-/- early gastrulating mouse embryos.

The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and Otx2(-/-) 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation.

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri.

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.

[DNA chip technology]. / La technologie des puces à ADN.

DNA chip technology has greatly evolved over the last decade and, associated with complete genome sequencing, is in the process of introducing a revolution into biological research. It is providing new and unique tools for studying emerging diseases outbreaks and epidemics. Nevertheless, microbiological surveillance, medical diagnosis, and field work involve a number of difficulties for which these new techniques have not yet been validated. Currently available chips are still limited in their application, but offer a powerful and economical alternative to former methods and will undoubtedly offer a range of unexplored applications in coming years.

The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector.

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.

Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression.

Bacillus subtilis grows in the absence of oxygen using nitrate ammonification and various fermentation processes. Lactate, acetate, and 2,3-butanediol were identified in the growth medium as the major anaerobic fermentation products by using high-performance liquid chromatography. Lactate formation was found to be dependent on the lctEP locus, encoding lactate dehydrogenase and a putative lactate permease. Mutation of lctE results in drastically reduced anaerobic growth independent of the presence of alternative electron acceptors, indicating the importance of NADH reoxidation by lactate dehydrogenase for the overall anaerobic energy metabolism. Anaerobic formation of 2,3-butanediol via acetoin involves acetolactate synthase and decarboxylase encoded by the alsSD operon. Mutation of alsSD has no significant effect on anaerobic growth. Anaerobic acetate synthesis from acetyl coenzyme A requires phosphotransacetylase encoded by pta. Similar to the case for lctEP, mutation of pta significantly reduces anaerobic fermentative and respiratory growth. The expression of both lctEP and alsSD is strongly induced under anaerobic conditions. Anaerobic lctEP and alsSD induction was found to be partially dependent on the gene encoding the redox regulator Fnr. The observed fnr dependence might be the result of Fnr-induced arfM (ywiD) transcription and subsequent lctEP and alsSD activation by the regulator ArfM (YwiD). The two-component regulatory system encoded by resDE is also involved in anaerobic lctEP induction. No direct resDE influence on the redox regulation of alsSD was observed. The alternative electron acceptor nitrate represses anaerobic lctEP and alsSD transcription. Nitrate repression requires resDE- and fnr-dependent expression of narGHJI, encoding respiratory nitrate reductase. The gene alsR, encoding a regulator potentially responding to changes of the intracellular pH and to acetate, is essential for anaerobic lctEP and alsSD expression. In agreement with its known aerobic function, no obvious oxygen- or nitrate-dependent pta regulation was observed. A model for the regulation of the anaerobic fermentation genes in B. subtilis is proposed.

Reprasentative Studie zur Verteilung schizophrener Patienten auf medizinische Versorgungseinrichtungen in Deutschland.

On the basis of a representative paper and pencil survey with office based psychiatrists, neurologists, psychiatric out-patient clinics and psychiatric wards/hospitals it is shown that in Germany in 1997 about 136,000 adult patients with diagnosed schizophrenia (F20 according to ICD 10) are attended by specialists. 75.4% of these patients are in attention of office based specialists, and 21.3% are attended by out-patient clinics. According to the details of psychiatric wards and hospitals, 3.3% of the patients are under long term (> 1 year) medical attention at hospitals. The amount of schizophrenia patients being under attention of specialists corresponds to 0.21% of the German population > 18 years. For patients who receive out-patient treatment it is shown that they are under medical attention at hospitals for average 22 days per year, and for 3.4 days they are under medical attention at rehabilitation centers.

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis.

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.