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Axillary brachial plexus block under neurostimulation is commonly used for upper limb surgery, but it is sometimes recognized as an uncomfortable technique, with most patients identifying electrical stimulation as an unpleasant moment. Ultrasound-guided regional anaesthesia, which becomes an increasingly popular technique, does not require electrical stimulation and then should theoretically improve axillary block placement comfort. The aim of this study was to compare the comfort of the patients during axillary block placement with neurostimulation and ultrasound guidance using either the out-of-plane or the in-plane approach.Consecutive patients were prospectively enrolled in three equal groups: neurostimulation, ultrasound out-of-plane and ultrasound in-plane approaches. A score was used to measure the comfort of the patients during axillary blocks placement. This score included three criteria: maximum pain intensity perceived during block placement measured using a visual analogue scale (0, no pain and 100, maximal or worse imaginable pain), the number of unpleasant events declared by the patients and the satisfaction of the patient (unsatisfied, acceptable, satisfied, very satisfied). The comfort score was calculated as the sum of each criterion, which was attributed a value of 0 or 1: visual analogue scale (or=30/100, 1;30/100, 0), number of unpleasant events (0, 1;or=1, 0) and satisfaction (satisfied or very satisfied, 1; acceptable or unsatisfied, 0). Procedures of axillary blocks placement resulting in a comfort score of 3 and 2 were arbitrary considered as very comfortable and comfortable, respectively. Success rate of axillary blocks, time to perform block and complications related to procedures were noted.One hundred and twenty patients were included. In the ultrasound out-of-plane group, 55% (22/40) and 25% (10/40) of the procedures were very comfortable and comfortable as compared with 32% (13/40, P0.05) and 20% (8/40, P0.01) in the ultrasound in-plane group and 25% (10/40, P0.01) and 8% (3/40, P0.01) in the neurostimulation group, respectively. Duration of axillary placement was significantly smaller in the ultrasound out-of-plane group as compared with that of in-plane approaches (P0.05) and neurostimulation (P0.01).The present study showed that the ultrasound approaches were less painful and more comfortable than neurostimulation to place axillary blocks. We also showed that, although pain intensity resulting from blocks placement was similar with the ultrasound approaches, very comfortable procedures were more frequent with the out-of-plane than with the in-plane approach.

Background & Aims Little is known about the magnitude of the risk of anal and rectal cancer in patients with anal and/or perineal Crohn's disease. We aimed to assess the risk of anal and rectal cancer in patients with Crohn's perianal disease followed up in the Cancers Et Surrisque Associe aux Maladies Inflammatoires Intestinales En France (CESAME) cohort. Methods We collected data from 19,486 patients with inflammatory bowel disease (IBD) enrolled in the observational CESAME study in France, from May 2004 through June 2005; 14.9% of participants had past or current anal and/or perianal Crohn's disease. Subjects were followed up for a median time of 35 months (interquartile range, 29–40 mo). To identify risk factors for anal cancer in the total CESAME population, we performed a case-control study in which participants were matched for age and sex. Results Among the total IBD population, 8 patients developed anal cancer and 14 patients developed rectal cancer. In the subgroup of 2911 patients with past or current anal and/or perianal Crohn's lesions at cohort entry, 2 developed anal squamous-cell carcinoma, 3 developed perianal fistula–related adenocarcinoma, and 6 developed rectal cancer. The corresponding incidence rates were 0.26 per 1000 patient-years for anal squamous-cell carcinoma, 0.38 per 1000 patient-years for perianal fistula–related adenocarcinoma, and 0.77 per 1000 patient-years for rectal cancer. Among the 16,575 patients with ulcerative colitis or Crohn's disease without anal or perianal lesions, the incidence rate of anal cancer was 0.08 per 1000 patient-years and of rectal cancer was 0.21 per 1000 patient-years. Among factors tested by univariate conditional regression (IBD subtype, disease duration, exposure to immune-suppressive therapy, presence of past or current anal and/or perianal lesions), the presence of past or current anal and/or perianal lesions at cohort entry was the only factor significantly associated with development of anal cancer (odds ratio, 11.2; 95% CI, 1.18-551.51; P = .03). Conclusions In an analysis of data from the CESAME cohort in France, patients with anal and/or perianal Crohn's disease have a high risk of anal cancer, including perianal fistula–related cancer, and a high risk of rectal cancer.

To further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine Mycobacterium bovis BCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of Mycobacterium bovis and M. bovis BCG. SNPs were assayed in M. bovis isolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent M. bovis strains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent M. bovis strains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent M. bovis strains and the attenuated BCG strain that will underpin future functional analyses.

Background and Objectives: We have compared ultrasound characteristics of spread during infraclavicular brachial-plexus blocks by use of electrically evoked radial-nerve- or median-nerve-type distal motor responses to guide the injection of 30 mL of 1.5% mepivacaine. Methods: Consecutive patients who required surgery distal to the upper arm were prospectively included in this study. With radial- or median-evoked distal motor response at a stimulating current intensity of less than 0.5 mA, patients were distributed into 2 equal groups. An independent investigator blinded to the evoked response described ultrasound characteristics of the spread of local anesthetic and assessed block quality 30 minutes after placement. A quality diffusion score proportional to the extent and intensity of spread around the axillary artery was used, and dynamic movements during injection were noted. Results: Thirty-two patients were included. With radial-nerve-type motor response, the success rate of infraclavicular plexus block was 100%, but 3 supplemental axillary blocks were requested with median-nerve-type motor response. Quality diffusion scores were significantly higher with radial-nerve-type as compared with median-nerve-type motor response (P = .03). Injection after radial-nerve-type motor response resulted in a typical and reproducible ultrasound feature of posterior local-anesthetic spread associated with medial and upper movement of the axillary artery. With median-nerve-type motor response, failed blocks were associated with a specific posterior displacement of the axillary artery that resulted from superficial spread. Conclusion: We have demonstrated that as compared with median-nerve-type motor response, injection performed after a radial-nerve-type motor response promoted reproducible and remarkable ultrasound spread characteristics associated with complete sensory block of the 3 cords at 30 minutes.

In the axillary fossa, the musculocutaneous nerve (MC) is generally distant from the axillary artery and from the other brachial plexus nerves. In that way, MC requires a specific block. We observed that the location of MC is influenced by the position of the patient's arm and shoulder. Abduction of the shoulder significantly reduced the distance between the MC and the axillary artery. This change in the location of the MC is probably due to the moving of the nerve because of muscle rearrangements and the ability to achieve better proximity of the probe in the axillary fossae.

We compared the success rate of single-injection infraclavicular plexus block by using electrically evoked radial, ulnar, or median nerve-type distal motor response to guide the injection of local anesthetic.Consecutive patients requiring surgery distal to the upper arm were prospectively included in this study over a 6-month period. No search for predetermined distal motor responses was performed. The first qualifying distal motor response evoked for a stimulating current intensity of0.5 mA distributed patients into 3 groups of patients. The study was continued until 3 groups of 60 patients were fulfilled. Twenty to 25 minutes after the injection of 30 mL of 1.5% mepivacaine, blinded evaluation of block quality was performed. A successful block was defined by the presence of a complete sensory block of the 5 major nerve distal distributions of the arm.Five hundred patients were included. The first evoked distal motor response was of radial, median, and ulnar nerve type in 46% (n = 230), 42% (n = 210), and 12% (n = 60) cases, respectively. The success rate of the infraclavicular plexus block was significantly higher when the injection was performed on a radial nerve-type response (90%) as compared with the median (74%) or ulnar (68%) nerve distal motor response. Intraoperative sedation and general anesthesia were not needed. None of the patients experienced specific complications.We showed that evoked distal motor response influenced the success rate of single-injection infraclavicular plexus block. The highest success rate was obtained when injection was performed after radial nerve-type motor response.

The small size of their genomes made bacterial ideal model organisms for the emerging field of genomics. Elucidating the genome sequences of mycobacteria was particularly attractive owing to the difficulties inherent in their manipulation. The slow growth rate, clumping, and requirement for category III containment make manipulation of Mycobacterium tuberculosis-complex strains laborious. M. leprae presents even greater problems as it has resisted all attempts at axenic culture. Availability of genome sequence data promised to accelerate our knowledge of the fundamental biology of these organisms, and to offer clues to the basis for their virulence, tropism and persistence in the host. This article will focus on what the genome sequences of M. tuberculosis and M. leprae have taught us about these pathogens, and how comparative genomics has exposed some of the fundamental differences between the species.

Population subgroups of the African malaria vector Anopheles gambiae have not been comprehensively characterized owing to the lack of unbiased sampling methods. In the arid savanna zone of West Africa, where potential oviposition sites are scarce, widespread collection from larval pools in the peridomestic human habitat yielded a comprehensive genetic survey of local A. gambiae population subgroups, independent of adult resting behavior and ecological preference. A previously unknown subgroup of exophilic A. gambiae is sympatric with the known endophilic A. gambiae in this region. The exophilic subgroup is abundant, lacks differentiation into M and S molecular forms, and is highly susceptible to infection with wild Plasmodium falciparum. These findings might have implications for the epidemiology of malaria transmission and control.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.

On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine.

Novel bioinformatics routines have been used to provide a more detailed definition of the proteome of Mycobacterium tuberculosis H37Rv. Over half of the current proteins result from gene duplication or domain shuffling events while one-sixth show no similarity to polypeptides described in other organisms. Prominent among the genes that appear to have been duplicated on numerous occasions are those involved in fatty acid metabolism, regulation of gene expression, and the unusually glycine-rich PE and PPE proteins. Protein similarity analysis, coupled with inspection of the genetic neighbourhood, was used to explore possible functional relatedness. This uncovered four large mce operons whose proteins may mediate initial interactions between the tubercle bacillus and host cells, together with a cluster of genes that might encode components of a structure required for secretion of ESAT-6 like proteins. Close linkage of the mmpL genes, encoding large membrane proteins, with those required for fatty acid metabolism suggests involvement in lipid transport. Compared to free-living bacteria, M. tuberculosis has a significantly smaller transport protein repertoire and this may reflect its intracellular lifestyle.

Detailed formulations are presented for an EHD model that studies all the engine main bearings, considering the bearings, housing and crankshaft elastic deformations in connection with the hydrodynamic pressure fields as well as the quasi-static loads balance for this complex kinematic system. The calculation process uses the finite element method and the Newton-Raphson method for the numerical analysis. The elastic crankshaft behavior is quantified by a standard stiffness matrix. With two structural analysis steps, a condensed housing stiffness matrix is obtained to reduce the computing time. The transient evolution of the cavitation area is evaluated with Murty’s algorithm. This model is utilized to study a four cylinder inline engine.

Leprosy, a chronic human disease with potentially debilitating neurological consequences, results from infection with Mycobacterium leprae . This unculturable pathogen has undergone extensive reductive evolution, with half of its genome now occupied by pseudogenes. Using comparative genomics, we demonstrated that all extant cases of leprosy are attributable to a single clone whose dissemination worldwide can be retraced from analysis of very rare single-nucleotide polymorphisms. The disease seems to have originated in Eastern Africa or the Near East and spread with successive human migrations. Europeans or North Africans introduced leprosy into West Africa and the Americas within the past 500 years.

The Ocean and Land Colour Imager (OLCI) is a high accuracy visible spectral imager selected as optical payload for the Sentinel 3 component of the GMES mission, to provide climatological data continuity with the previous ESA Envisat missions. OLCI is based on the very successful opto-mechanical and imaging design of MERIS. The instrument is a quasi-autonomous, self contained, visible-NIR push-broom imaging spectrometer and incorporates significant improvements when compared to MERIS. The paper highlights the technical and programmatic challenges of the project, first results from the EM test activities and a projected flight model performance summary.

International audience; Functional studies have demonstrated a role for the Anopheles gambiae APL1A gene in resistance against the human malaria parasite, Plasmodium falciparum. Here, we exhaustively characterize the structure of the APL1 locus and show that three structurally different APL1A alleles segregate in the Ngousso colony. Genetic association combined with RNAi-mediated gene silencing revealed that APL1A alleles display distinct protective profiles against P. falciparum. One APL1A allele is sufficient to explain the protective phenotype of APL1A observed in silencing experiments. Epitope-tagged APL1A isoforms expressed in an in vitro hemocyte-like cell system showed that under assay conditions, the most protective APL1A isoform (APL1A(2)) localizes within large cytoplasmic vesicles, is not constitutively secreted, and forms only one protein complex, while a less protective isoform (APL1A(1)) is constitutively secreted in at least two protein complexes. The tested alleles are identical to natural variants in the wild A. gambiae population, suggesting that APL1A genetic variation could be a factor underlying natural heterogeneity of vector susceptibility to P. falciparum.

A recombinant lambda phage was identified in a Clostridium perfringens genomic library by means of its ability to hydrolyse the fluorescent substrate 4-methyl-umbelliferyl-beta-D-glucosaminide, isolated and shown to encode an endo-beta-N-acetylglucosaminidase. This enzyme, NagH, is also known as hyaluronidase, or Mu toxin, a putative virulence factor which is likely to act on connective tissue during gas gangrene. Nucleotide sequence analysis allowed the primary structure to be deduced and showed hyaluronidase to be a large exported protein of 114,392 Daltons and an enzyme of this size, endowed with the corresponding activities, was partially purified from C. perfringens. Hyaluronidase seems to be organised into two domains, an N-terminal region comprising 700 amino acids bearing the active site and a 300-residue C-terminal segment, containing three copies of an extended motif. Two other reading frames, linked to nagH, also appear to encode proteins with sugar-binding motifs.

Objectives Ultrasound-guided regional anesthesia is commonly used for block placement. At present, the risk of cross contamination from probes is not well documented. To avoid transmission of infectious agents, several methods have been used for probe disinfection and protection. The aim of this study was to evaluate the antibacterial efficacy of a new high-level disinfection method based on ultraviolet C (UV-C) light under routine conditions after block placement with an unprotected probe. Methods The study was after approval by the local Ethics Committee. In the first part of the study, 15 ultrasound probes were exposed to a large inoculum of 3 bacteria. Ultraviolet C disinfection consisted of cleaning the probe with dry and disinfectant-impregnated paper followed by a 90-second UV-C disinfection cycle in a decontamination chamber. A protocol was established to retrieve the probe with sterile gloves after opening the door of the chamber. In the second part, 50 blocks were placed with ultrasound-guided regional anesthesia. The skin was first prepared with an antiseptic solution, and sterile gel was applied; no covers were used to protect the probes. The blocks were then disinfected with UV-C light. Bacteriologic samples were collected before and after the UV-C method and inoculated on chocolate agar plates. Results During the first part of the study, all probes were infected after inoculation (>150 colony-forming units) but were considered sterile (

Background: Because poor echogenicity of the needle remains a safety issue, we decided to analyze the learning process of the hydrolocalization technique (Hloc) performed to continuously identify needle-tip anatomical position during many ultrasound-guided regional anesthesia procedures. Methods: Ten senior anesthesiologists naive to the Hloc agreed to participate in the study. They were requested to perform 40 out-of-plane (OOP) approach ultrasound-guided axillary blocks (AB) each using the Hloc. The Hloc, which is a needle-tip localization principle, was performed by means of repetitive injections of a small amount of a local anesthetic solution (0.5–1 ml) under an ultrasound beam. Details of the learning process and skill acquisition of the Hloc were derived from the following parameters: the duration of block placement, a measure of the perceived difficulty of needle-tip visualization, a measure of block placement difficulty, and the amount of local anesthetics solution required for the technique. Results: Four hundred ABs were performed. The success rate of an ultrasound-guided AB was 98%. The Hloc was successful in all patients. Skill acquisition over time of the Hloc was associated with a significant reduction of both the duration and the perceived difficulty of ABs placement. Apprenticeship data revealed that 20 blocks were required to successfully place AB within 5 min in most cases using the Hloc. Conclusion: The Hloc performed during the OOP approach of ultrasound-guided regional anesthesia is a simple technique with a relatively short learning process feasible for efficient placement of ABs.

To further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine Mycobacterium bovis BCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of Mycobacterium bovis and M. bovis BCG. SNPs were assayed in M. bovis isolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent M. bovis strains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent M. bovis strains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent M. bovis strains and the attenuated BCG strain that will underpin future functional analyses.

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra “knock-ins” that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis., Author Summary Mycobacterium tuberculosis, the causative agent of human tuberculosis is an extremely successful human pathogen in spite of its lack of classical virulence factors, such as toxins. The pathogenesis of this bacterium is closely linked with its ability to circumvent destruction by the host cell, which depends on a large variety of mycobacterial lipids and secreted proteins. Genome comparison of fully virulent strains with closely related, but attenuated strains that have lost their ability to cause disease is a powerful approach to identify factors contributing to mycobacterial virulence. In this study we have compared the virulent paradigm strain of tuberculosis research M. tuberculosis H37Rv, with the widely used, attenuated laboratory variant, H37Ra. This analysis, combined with gene complementation studies, has identified a mutation in the two-component system regulator PhoP that impacts on the secretion and T cell recognition of the 6-kD early-secreted antigenic target ESAT-6. In previous studies, both PhoP and the ESAT-6 secretion system ESX-1 have been identified independently as major virulence determinants of M. tuberculosis, so the finding that their action is interconnected opens exciting new insights and perspectives into the virulence regulation of tubercle bacilli.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette–Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2 , is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding σ-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

In silico analysis reveals that most protective antigens expressed by the antituberculous vaccineMycobacterium bovisBCG (BCG) are conserved inM. avium, supporting the hypothesis that exposure to environmental mycobacteria generates cross-reactive immune responses blocking BCG activity. We investigated the impact of sensitization withM. avium,M. scrofulaceum, orM. vaccaeon the protective efficacy of a recombinant BCG strain expressing RD1 antigens (BCG::RD1), using a mouse model of experimental tuberculosis (TB). No evidence that the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were expressed by these environmental strains could be demonstrated by Western blot analysis. Mice sensitized with each of these strains did not prime cellular immune responses cross-reacting with the immunodominant ESAT-6. Importantly, clearance of BCG::RD1 from the lungs and spleens of mice exposed to each of the environmental strains before vaccination was minimal compared to that of BCG. In mice sensitized withM. avium, increased persistence of BCG::RD1 correlated with stronger antimycobacterial gamma interferon responses and enhanced protection against aerosol infection withM. tuberculosis, compared to BCG. In contrast, animals exposed toM. scrofulaceumorM. vaccaeprior to vaccination with BCG or BCG::RD1 were better protected against TB than were the unsensitized controls. Our results suggest that the inhibitory effect of environmental mycobacteria on the protective efficacy of BCG depends critically on the extent of cross-recognition of antigens shared with the vaccine. In hosts sensitized withM. avium, potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 may allow this recombinant vaccine to overcome preexisting antimycobacterial responses.

The 174-kb virulence plasmid pMUM001 inMycobacterium ulceransepidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation ofM. ulceransAgy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverseM. ulceransstrains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in allM. ulceransstrains. Physical mapping of these plasmids revealed that likeM. ulceransAgy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis thatM. ulceransdiverged from the closely related organismMycobacterium marinumby acquiring pMUM. Our results suggest that pMUM is a defining characteristic ofM. ulceransbut that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium , and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis , Mycobacterium africanum , Mycobacterium canettii , Mycobacterium microti , and Mycobacterium bovis . This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosi s strains can be divided into ancestral and “modern” strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum , M. microti , and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis , the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum→M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

Outdoor-resting (exophilic) mosquitoes have been little studied because of the lack of efficient outdoor sampling methods. Pools of water for female mosquito oviposition are a limiting resource, and therefore collecting mosquitoes from larval pools should sample all members of the population

The integrated map of the Mycobacterium leprae genome unveiled for the first time the genomic organization of this obligate intracellular parasite. Selected cosmid clones, isolated from a genomic library created in the cosmid vector Lorist6, were identified as representing nearly the complete genome and were subsequently used in the M. leprae genome sequencing project. Now a new version of the integrated map of M. leprae can be presented, combining the mapping results from the Lorist6 cosmids with data obtained from a second genomic library constructed in an Escherichia coli-mycobacterium shuttle cosmid, pYUB18. More than 98% of the M. leprae genome is now covered by overlapping large insert genomic clones representing a renewable source of well defined DNA segments and a powerful tool for functional genomics.

Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.

To achieve the quantum leap in understanding required to overcome two major human diseases, leprosy and tuberculosis, systematic and comparative genome analysis has been undertaken. New insight into the biology of their causative agents has been obtained and the principle findings are reported here.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

International audience

The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cereus (PC-PLC), for which a high-resolution crystal structure is available. This structural information was used as the basis of a site-directed mutagenesis strategy in which critical amino acid residues of alpha-toxin involved in zinc binding, interaction with substrate, or catalysis were replaced. Biochemical studies with the corresponding toxin variants indicate that there is probably a single active site endowed with lecithinase, sphingomyelinase, and hemolytic activities. By using a highly purified variant in which the catalytic aspartate residue at position 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platelet aggregation in vitro.

International audience; The physical map of the 3.6-megabase chromosome of Clostridium perfringens CPN50 was extended by positioning sites for the endonucleases SfiI and I-CeuI, and in parallel, the gene map was expanded by using a genome scanning strategy. This involved the cloning and sequencing of random chromosomal fragments, identification of the functions of the putative genes by database searches, and then hybridization analysis. The current gene map comprises almost 100 markers, many of which encode housekeeping functions while others are involved in sporulation or pathogenesis. Strikingly, most of the virulence genes were found to be confined to a 1,200-kb segment of the chromosome near oriC, while the pleiotropic regulatory locus, virRS, was situated toward the putative replication terminus. A comparison of the gene maps of three endospore-forming bacilli, C. perfringens, Clostridium beijerinckii, and Bacillus subtilis, revealed a similar order and distribution of key sporulation and heat shock genes which might reflect an ancient evolutionary relationship.

In the 1970s, Ionesco and Bouanchaud (1973) reported the first demonstration for a dostridial species that bacteriocin production was associated with the presence of a small plasmid. It was later shown that Clostridium perfringens CPN50 synthesized large quantities of the bacteriocin BCN5 after irradiation with ultraviolet light and that this bactericidal protein was secreted into the medium (Ionesco et al., 1974). Subsequent genetic studies revealed that the bacteriocinogenic plasmid, pIP404, was about 10 kb in size and could be mobilized by conjugation by certain cryptic plasmids commonly found in C. perfringens (Brefort et al., 1977). Several years later, pIP404 was adopted by our laboratory as a model system for studying DNA damage-inducible genes in the Clostridia and as a source of material for vector construction.

The nucleotide sequence of cosmid B1790, carrying the Rif-Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the beta and beta' subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so-called ABC family of ATP-binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages.

Nat. Genet. 41, 1282–1289 (2009); published online 1 November 2009; corrected after print 5 February 2010. In the version of this article initially published 1 November 2009, Ali Khamesipour was misspelled in the author list. The error has been corrected in the HTML and PDF versions of the article.

The bacteriocinogenic plasmid pIP404 from Clostridium perfringens was isolated and cloned in Escherichia coli, and its physical map was deduced. Expression of the bcn gene, encoding bacteriocin BCN5, is inducible by UV irradiation of C. perfringens and thus resembles the SOS-regulated bacteriocin genes of enteric bacteria. The location of bcn on pIP404 was established by a dot-blot procedure, using specific hybridization probes to analyze mRNA samples from induced and uninduced cultures. From the nucleotide sequence of its gene, the molecular weight of BCN5 was deduced to be 96,591, and a protein of this size was secreted by bacteriocin-producing cultures of C. perfringens. The primary structure of the protein suggests that it may function as an ionophore, since a hydrophobic domain, resembling those of the ionophoric colicins, is present at the COOH terminus. No bacteriocin activity could be detected in E. coli harboring plasmids bearing the bcn gene, even when the transcriptional and translational signals were replaced by those of lacZ. A possible explanation may be found in the unusual codon usage of the adenine-thymine-rich bcn gene, as this shows a preference for codons with a high adenine-plus-thymine content, especially in the wobble position. Many of the frequently used codons correspond to those recognized by minor tRNA species in E. coli. Consequently, bcn expression might be limited by tRNA availability in this bacterium.

The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined. The plasmid genome comprises 10,207 bp and has a dA + dT content of 75%. Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified. The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content. Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.

Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as pIP404 which, in addition to a set of UV-inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site-specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram-negative and Gram-positive bacteria as well as enzymes involved in site-specific DNA inversion. A likely role for the RES protein would be to stabilize pIP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site-specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Streptococcus faecalis.