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Accueil > Départements > Microbiologie > Anciennes équipes du département > Michael DUBOW : Génomique et Biodiversité microbienne des biofilms

Publications de l’équipe


  • A. Briquet, R. Vong, J. - B. Roseau, E. Javelle, N. Cazes, F. Rivière, M. Aletti, M. - P. Otto, C. Ficko, S. Duron, M. Fabre, C. Pourcel, F. Simon, et C. Soler, « Clinical features of Mycobacterium canettii infection: a retrospective study of 20 cases among French soldiers and relatives », Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, févr. 2019.
    Résumé : Background: Mycobacterium canettii forms part of the Mycobacterium tuberculosis complex. M. canettii infections are mainly described in the Horn of Africa. The permanent presence of French soldiers in Djibouti raises the question of the risk of being infected with M. canettii. Our study aims to describe M. canettii infections among French military or their families between 1998 and 2015. Methods: This retrospective study relied on 3 sources of data: the reference centre for mycobacteria in the Biology Department at Percy military hospital in Paris, the French Military Center for Epidemiology and Public Health, and the scientific literature. After an exhaustive census of the strains, we studied the epidemiological data on 20 cases among French soldiers and their families. Results: 20 cases of M. canettii infections are reported, including 5 unpublished cases. Adenitis predominates (n = 15), especially in the cervico facial area and among children; one case was observed one month after dental care in Djibouti. The pulmonary forms were less frequent (n = 6) and 3 atypical forms are described. All patients had stayed in Djibouti. Conclusions: Cases of M. canettii infection among the French military consisted mainly of adenitis; disseminated forms were possible with immunodeficiency. Their evolution under specific treatments were comparable to tuberculosis. The presumed origin of the infection seemed to be environmental, possibly a water reservoir, and not due to human-to-human contagion.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • C. Essoh, J. - P. Vernadet, G. Vergnaud, A. Coulibaly, A. Kakou-N'Douba, A. S. - P. N'Guetta, G. Resch, et C. Pourcel, « Complete Genome Sequences of Five Acinetobacter baumannii Phages from Abidjan, Côte d'Ivoire », Microbiology Resource Announcements, vol. 8, nᵒ 1, janv. 2019.
    Résumé : Five bacteriophages of Acinetobacter baumannii were isolated from sewage water in Abidjan, Côte d'Ivoire. Phages Aci01-1, Aci02-2, and Aci05 belong to an unclassified genus of the Myoviridae family, with double-stranded DNA (dsDNA) genomes, whereas Aci07 and Aci08 belong to the Fri1virus genus of the Podoviridae family of phages.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • J. Lossouarn, A. Briet, E. Moncaut, S. Furlan, A. Bouteau, O. Son, M. Leroy, M. S. DuBow, F. Lecointe, P. Serror, et M. - A. Petit, « Enterococcus faecalis Countermeasures Defeat a Virulent Picovirinae Bacteriophage », Viruses, vol. 11, nᵒ 1, p. 48, janv. 2019.
    Résumé : Enterococcus faecalis is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain E. faecalis V583 to isolate a virulent phage belonging to the Picovirinae subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of E. faecalis tested—including V583—were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that E. faecalis V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected E. faecalis. The abiα gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10−4 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.
    Mots-clés : <i>Enterococcus</i>, abortive infection, adsorption, bacterial, encodes, Enterococcus, escherichia-coli, family, gene, identification, LGBMB, MICROBIO, phage abortive infection, prophage, protein, resistance mechanism, rhamnopolysaccharide, system.
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  • J. R. Osman, C. Regeard, C. Badel, G. Fernandes, et M. S. DuBow, « Variation of bacterial biodiversity from saline soils and estuary sediments present near the Mediterranean Sea coast of Camargue (France) », Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, vol. 112, nᵒ 3, p. 351-365, mars 2019.
    Résumé : Salinity is an important environmental factor influencing microbial community composition. To better understand this influence, we determined the bacterial communities present in 17 different sites of brackish sediment (underwater) and soil (surface) samples from the Camargue region (Rhone river delta) in southern France during the fall of 2013 and 2014 using pyrosequencing of the V3-V4 regions of the 16S rRNA genes amplified by PCR. This region is known for abundant flora and fauna and, though saline, 30% of rice consumed in France is grown here. We found that bacterial abundance in 1g of soil or sediment, calculated by qPCR, was higher in sediments than in surface soil samples. Members belonging to the Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes phyla dominated the bacterial communities of sediment samples, while members belonging to the Proteobacteria, Bacteroidetes, Gemmatimonadetes, Actinobacteria, Firmicutes and Acidobacteria phyla dominated the bacterial communities of the soil samples. The most abundant bacterial genera present in the saline sediments and soils from the Camargue belonged mostly to halophilic and sulphate reducing bacteria, suggesting that the Camargue may be a valuable system to investigate saline, yet agriculturally productive, sediment and soil microbial ecosystem.
    Mots-clés : ARCHEE, Bacterial biodiversity, community structure, de-giraud, diversity, gen. nov., Halophilic bacteria, hypersaline microbial mat, LGBMB, MICROBIO, molecular analysis, Pyrosequencing, rhone river delta, rice fields, rna, Salinity, temporal variation.


  • D. Couvin, A. Bernheim, C. Toffano-Nioche, M. Touchon, J. Michalik, B. Néron, E. P. C Rocha, G. Vergnaud, D. Gautheret, et C. Pourcel, « CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins », Nucleic Acids Research, mai 2018.
    Résumé : CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • L. Latino et C. Pourcel, « Recovery and Characterization of Bacteria Resisting Infection by Lytic Bacteriophage », Methods in Molecular Biology (Clifton, N.J.), vol. 1693, p. 85-98, 2018.
    Résumé : Bacteria and bacteriophages coexist and coevolve, bacteriophages being obligatory predators exerting an evolutionary pressure on their prey. Mechanisms in action vary depending on the bacterial genomic content and on the regulation of the bacteriophage cycle. To assess the multiplicity of bacterial genes involved in resistance as well as the changes in the bacteriophage interactions with the bacteria, it is necessary to isolate and investigate large numbers of independent resistant variants. Here we describe protocols that have been applied to the study of Pseudomonas aeruginosa and four of its virulent bacteriophages belonging to the Podoviridae and Myoviridae bacteriophage families. Mutations are identified using whole genome sequencing of resistant variants. Phenotypic analyses are performed to describe the changes conferred by the mutations.
    Mots-clés : Bacterial phenotype, Bacteriophages, Complementation, Genome sequencing, LGBMB, MICROBIO.

  • H. Liu, Y. Zhang, Z. Liu, J. Liu, Y. Hauck, J. Liu, H. Dong, J. Liu, X. Zhao, B. Lu, Y. Jiang, G. Vergnaud, C. Pourcel, et K. Wan, « Associations between Mycobacterium tuberculosis Beijing genotype and drug resistance to four first-line drugs: a survey in China », Frontiers of Medicine, vol. 12, nᵒ 1, p. 92-97, 2018.

  • J. R. Osman, G. Fernandes, C. Regeard, C. Jaubert, et M. S. DuBow, « Examination of the Bacterial Biodiversity of Coastal Eroded Surface Soils from the Padza de Dapani (Mayotte Island) », Geomicrobiology Journal, vol. 35, nᵒ 5, p. 355-365, 2018.
    Résumé : To better understand microbial populations present in atypical soil environments, and to discern any relations between these environments and their bacterial communities, we examined the “Padza de Dapani” on the island of Mayotte off the east coast of Africa. This area is not a true (hot) desert, but resembles one in many places due to extensive soil erosion. We collected surface soil samples from five different sites of the Padza de Dapani in Mayotte. We examined bacterial biodiversity using pyrosequencing of PCR-amplified 16S V1–V3 rDNA sequences from total extracted DNA. Our results show that in the acidic (pH 4.6–6), oligotrophic (organic carbon; 0.1–0.7 g/kg of soil) and mineralized (Fe: 18 g/100 g; Al: 12 g/100 g) Dapani Padza soil samples, members of the Actinobacteria and Proteobacteria phyla dominated the bacterial communities, as was also observed in samples from Saudi Arabia hot desert sands.Interestingly, members belonging to the genera Acinetobacter, Arthrobacter and Bacillus were also found to be very abundant in our samples. These were also seen in hot Asian deserts sand samples, such as those from the Gobi (Mongolia) and Taklamaken (China) deserts, thus possibly pointing to microbial populations characteristic of denuded soils.
    Mots-clés : 16S rRNA, Bacteria, biodiversity, LGBMB, MICROBIO, pyrosequencing, soil erosion.

  • X. Qiu, J. Xu, J. Guo, A. Yahia-Ammar, N. - I. Kapetanakis, I. Duroux-Richard, J. J. Unterluggauer, N. Golob-Schwarzl, C. Regeard, C. Uzan, S. Gouy, M. DuBow, J. Haybaeck, F. Apparailly, P. Busson, et N. Hildebrandt, « Advanced microRNA-based cancer diagnostics using amplified time-gated FRET », Chemical Science, vol. 9, nᵒ 42, p. 8046-8055, nov. 2018.
    Résumé : MicroRNAs (miRNAs) play an important role in cellular functions and in the development and progression of cancer. Precise quantification of endogenous miRNAs from different clinical patient and control samples combined with a one-to-one comparison to standard technologies is a challenging but necessary endeavor that is largely neglected by many emerging fluorescence technologies. Here, we present a simple, precise, sensitive, and specific ratiometric assay for absolute quantification of miRNAs. Isothermally amplified time-gated Forster resonance energy transfer (TG-FRET) between Tb donors and dye acceptors resulted in miRNA assays with single-nucleotide variant specificity and detection limits down to 4.2 +/- 0.5 attomoles. Quantification of miR-21 from human tissues and plasma samples revealed the relevance for breast and ovarian cancer diagnostics. Analysis of miR-132 and miR-146a from acute monocytic leukemia cells (THP-1) demonstrated the broad applicability to different miRNAs and other types of clinical samples. Direct comparison to the gold standard RT-qPCR showed advantages of amplified TG-FRET concerning precision and specificity when quantifying low concentrations of miRNAs as required for diagnostic applications. Our results demonstrate that a careful implementation of rolling circle amplification and TG-FRET into one straightforward nucleic acid detection method can significantly advance the possibilities of miRNA-based cancer diagnostics and research.
    Mots-clés : assay, B3S, isothermal amplification, LGBMB, MICROBIO, NANO, rolling circle amplification, strategy.

  • H. Salmi-Mani, G. Terreros, N. Barroca-Aubry, C. Aymes-Chodur, C. Regeard, et P. Roger, « Poly(ethylene terephthalate) films modified by UV-induced surface graft polymerization of vanillin derived monomer for antibacterial activity », European Polymer Journal, vol. 103, p. 51-58, juin 2018.
    Résumé : New antibacterial PET surfaces were developed from vanillin-derived biobased monomer. An easy one-step and high yielding synthesis of N-(4-hydroxy-3-methoxybenzyl)-acrylamide monomer was successfully achieved. PET was modified by a two-step procedure: Type II photoinitiator was first grafted through a PET aminolysis with N,N-diethylethylenediamine, then the photopolymerization of the biobased acrylamide monomer was performed according to a “grafting from” technique. PET surface modifications were characterized by XPS and UV–visible spectroscopies, as well as water contact angle measurements. Finally, antiadhesion biotests were conducted to evaluate the potential antibacterial performances of the modified surfaces against gram-positive (Rhodococcus wratislaviensis and Staphylococcus aureus) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains.
    Mots-clés : LGBMB, MICROBIO.

  • G. Vergnaud, Y. Hauck, D. Christiany, B. Daoud, C. Pourcel, I. Jacques, A. Cloeckaert, et M. S. Zygmunt, « Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974-2006 », Frontiers in Microbiology, vol. 9, p. 1545, 2018.
    Résumé : Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.
    Mots-clés : animal, Brucella, genotyping, human, LGBMB, MICROBIO, MLVA, population structure.

  • G. Vergnaud, C. Midoux, Y. Blouin, M. Bourkaltseva, V. Krylov, et C. Pourcel, « Transposition Behavior Revealed by High-Resolution Description of Pseudomonas Aeruginosa Saltovirus Integration Sites », Viruses, vol. 10, nᵒ 5, 2018.
    Résumé : Transposable phages, also called saltoviruses, of which the Escherichia coli phage Mu is the reference, are temperate phages that multiply their genome through replicative transposition at multiple sites in their host chromosome. The viral genome is packaged together with host DNA at both ends. In the present work, genome sequencing of three Pseudomonas aeruginosa transposable phages, HW12, 2P1, and Ab30, incidentally gave us access to the location of thousands of replicative integration sites and revealed the existence of a variable number of hotspots. Taking advantage of deep sequencing, we then designed an experiment to study 13,000,000 transposon integration sites of bacteriophage Ab30. The investigation revealed the presence of 42 transposition hotspots adjacent to bacterial interspersed mosaic elements (BIME) accounting for 5% of all transposition sites. The rest of the sites appeared widely distributed with the exception of coldspots associated with low G-C content segments, including the putative O-antigen biosynthesis cluster. Surprisingly, 0.4% of the transposition events occurred in a copy of the phage genome itself, indicating that the previously described immunity against such events is slightly leaky. This observation allowed drawing an image of the phage chromosome supercoiling into four loops.
    Mots-clés : chromosomal domain, deep sequencing, hotspots, imaging, LGBMB, MICROBIO, supercoiling, transposable phages, transposon integration.


  • S. Al Dahouk, S. Köhler, A. Occhialini, M. P. Jiménez de Bagüés, J. A. Hammerl, T. Eisenberg, G. Vergnaud, A. Cloeckaert, M. S. Zygmunt, A. M. Whatmore, F. Melzer, K. P. Drees, J. T. Foster, A. R. Wattam, et H. C. Scholz, « Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts », Scientific Reports, vol. 7, p. 44420, mars 2017.
    Résumé : Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.
    Mots-clés : LGBMB, MICROBIO.

  • K. Bangpanwimon, J. Sottisuporn, P. Mittraparp-Arthorn, W. Ueaphatthanaphanich, A. Rattanasupar, C. Pourcel, et V. Vuddhakul, « Correction to: CRISPR-like sequences in Helicobacter pylori and application in genotyping », Gut Pathogens, vol. 9, p. 72, 2017.
    Résumé : [This corrects the article DOI: 10.1186/s13099-017-0215-8.].
    Mots-clés : LGBMB, MICROBIO.

  • K. Bangpanwimon, J. Sottisuporn, P. Mittraparp-Arthorn, W. Ueaphatthanaphanich, A. Rattanasupar, C. Pourcel, et V. Vuddhakul, « CRISPR-like sequences in Helicobacter pylori and application in genotyping », Gut Pathogens, vol. 9, p. 65, 2017.
    Résumé : Background: Many bacteria and archaea possess a defense system called clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas system) against invaders such as phages or plasmids. This system has not been demonstrated in Helicobacter pylori. The numbers of spacer in CRISPR array differ among bacterial strains and can be used as a genetic marker for bacterial typing. Results: A total of 36 H. pylori isolates were collected from patients in three hospitals located in the central (PBH) and southern (SKH) regions of Thailand. It is of interest that CRISPR-like sequences of this bacterium were detected in vlpC encoded for VacA-like protein C. Virulence genes were investigated and the most pathogenic genotype (cagA vacA s1m1) was detected in 17 out of 29 (58.6%) isolates from PBH and 5 out of 7 (71.4%) from SKH. vapD gene was identified in each one isolate from PBH and SKH. CRISPR-like sequences and virulence genes of 20 isolates of H. pylori obtained in this study were analyzed and CRISPR-virulence typing was constructed and compared to profiles obtained by the random amplification of polymorphic DNA (RAPD) technique. The discriminatory power (DI) of CRISPR-virulence typing was not different from RAPD typing. Conclusion: CRISPR-virulence typing in H. pylori is easy and reliable for epidemiology and can be used for inter-laboratory interpretation.
    Mots-clés : CRISPR-like sequences, CRISPR-virulence typing, Helicobacter pylori, LGBMB, MICROBIO, Orphan CRISPR array, vacA-like gene, vlpC gene.

  • R. Chouari, M. Leonard, M. Bouali, S. Guermazi, N. Rahli, I. Zrafi, L. Morin, et A. Sghir, « Eukaryotic molecular diversity at different steps of the wastewater treatment plant process reveals more phylogenetic novel lineages », World Journal of Microbiology & Biotechnology, vol. 33, nᵒ 3, p. 44, mars 2017.
    Résumé : Wastewater microbiota represents important actors of organic depollution. Nowadays, some species used as bioindicators of the effluent quality are still identified by microscopy. In the present study, we investigated eukaryotic diversity at the different steps of the treatment process of a wastewater treatment plant (aerobic, anaerobic, clarifier basins and anaerobic digester) using the 18S rRNA gene sequencing approach. Of the 1519 analysed sequences, we identified 160 operational taxonomic units. Interestingly, 56.9% of the phylotypes were assigned to novel phylogenetic molecular species since they show <97% sequence identity with their nearest affiliated representative within public databases. Peritrichia ciliates were the most predominant group, with Epistylis as the most common genus. Although anaerobic, the digester appears to harbor many unclassified phylotypes of protozoa species. Novel lineages such as LKM11 and LKM118 were widely represented in the digester. Diversity values given by Shannon indexes show that the clarifier is the most diversified. This work will help designing molecular tools that are fast, reliable, and reproducible for monitoring wastewater depollution and studying phylogenetic relationships among the wonderful world of protists within this anthropogenic ecosystem.
    Mots-clés : 18S rRNA gene, Activated sludge, Ciliates, Cryptomycota, LGBMB, LKM118, MICROBIO, Wastewater microbiota.

  • K. Gloux, M. Guillemet, C. Soler, C. Morvan, D. Halpern, C. Pourcel, H. Vu Thien, G. Lamberet, et A. Gruss, « Clinical relevance of FASII bypass in Staphylococcus aureus », Antimicrobial Agents and Chemotherapy, févr. 2017.
    Résumé : The need for new antimicrobials to treat bacterial infections has led to the use of fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host, and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, non-culturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of non-culturable variants.
    Mots-clés : LGBMB, MICROBIO.

  • L. Latino, M. Caroff, et C. Pourcel, « Fine structure analysis of lipopolysaccharides in bacteriophage-resistant Pseudomonas aeruginosa PAO1 mutants », Microbiology (Reading, England), vol. 163, nᵒ 6, p. 848-855, juin 2017.
    Résumé : Pseudomonas aeruginosa lipopolysaccharides (LPS) serve as primary receptors for many bacteriophages and, consequently, their biosynthesis is frequently affected in phage-resistant mutants. We previously isolated phage-resistant PAO1 mutants using three different phages, and showed that they were affected in the synthesis of LPS. Here we have investigated in detail the effect of mutations in seven genes involved in different steps of the production of core and oligosaccharide chains. The band profile of purified LPS was analysed by PAGE, and we further characterized the O-chains and core structures by MALDI mass spectrometry (MS). Mild LPS extraction conditions and native LPS MS analyses helped unveil lipid A molecular species with three phosphate residues in the close vicinity of the already highly charged inner-core region. No other MS direct analysis has allowed this peculiarity to be demonstrated for native lipid A high-molecular-weight molecular species, in normal growth conditions and without involving separation techniques. The present results shed light on the possible interactions between the phages and the LPS structures in the early phase of infection.
    Mots-clés : Bacteriophages, CAROFF, DIR, LGBMB, Lipopolysaccharides, Mass Spectrometry, MICROBIO, Mutation, Pseudomonas aeruginosa, Receptors, Virus.

  • J. R. Osman, G. Fernandes, et M. S. DuBow, « Bacterial diversity of the rhizosphere and nearby surface soil of rice (Oryza sativa) growing in the Camargue (France) », Rhizosphere, vol. 3, p. 112-122, 2017.
    Résumé : Bacterial communities present in the rhizosphere of different plants, including rice (Oryza sativa), play an essential key role in biogeochemical cycles, plant nutrition and disease biocontrol. The Camargue area of France, part of the Rhône river delta flowing into the Mediterranean Sea, is considered a saline ecosystem. In order to understand the soil bacterial ecology of the Camargue rice growing areas, we collected samples from six different sites, from the rhizosphere of two rice varieties (Arelate and Gageron) at late stages of growth, plus adjacent bulk soil samples, during 2013 and 2014. We used pyrosequencing of PCR amplified V3-V4 regions of the 16S rRNA gene from total extracted DNA to identify the bacterial communities present, and found that the principal bacterial phyla were composed of members belonging to the Proteobacteria, Acidobacteria, Chloroflexi, Bacteroidetes and Gemmatimonadetes phyla. While the relative abundance of these phyla did not differ between the rhizosphere and bulk soil samples, the abundances varied significantly among sites and between the years of collection. The most abundant bacterial groups at both the phylum and genus levels were similar to those found in other rice growing soils, though only one halophilic genus was present in significant numbers. Our work also suggests that the challenges of rice cultivation in proximity to relatively high-salt soils can likely be overcome by current irrigation and crop-rotation strategies.
    Mots-clés : Bacteria, Bacterial diversity, LGBMB, MICROBIO, Pyrosequencing, Rhizosphere.

  • C. Pourcel, C. Midoux, Y. Hauck, G. Vergnaud, et L. Latino, « Large Preferred Region for Packaging of Bacterial DNA by phiC725A, a Novel Pseudomonas aeruginosa F116-Like Bacteriophage », PloS One, vol. 12, nᵒ 1, p. e0169684, 2017.
    Résumé : Bacteriophage vB_PaeP_PAO1_phiC725A (short name phiC725A) was isolated following mitomycin C induction of C7-25, a clinical Pseudomonas aeruginosa strain carrying phiC725A as a prophage. The phiC725A genome sequence shows similarity to F116, a P. aeruginosa podovirus capable of generalized transduction. Likewise, phiC725A is a podovirus with long tail fibers. PhiC725A was able to lysogenize two additional P. aeruginosa strains in which it was maintained both as a prophage and in an episomal state. Investigation by deep sequencing showed that bacterial DNA carried inside phage particles originated predominantly from a 700-800kb region, immediately flanking the attL prophage insertion site, whether the phages were induced from a lysogen or recovered after infection. This indicates that during productive replication, recombination of phage genomes with the bacterial chromosome at the att site occurs occasionally, allowing packaging of adjacent bacterial DNA.
    Mots-clés : DNA, Bacterial, Gene Order, Genome, Bacterial, Genome, Viral, LGBMB, Lysogeny, MICROBIO, Pseudomonas aeruginosa, Pseudomonas Phages, Virion, Virus Assembly.

  • C. Pourcel, C. Midoux, G. Vergnaud, et L. Latino, « A carrier state is established in Pseudomonas aeruginosa by phage LeviOr01, a newly isolated ssRNA levivirus », The Journal of General Virology, août 2017.
    Résumé : ssRNA bacteriophages are very abundant but poorly studied, particularly in relation to their effect on bacterial evolution. We isolated a new Pseudomonas aeruginosa levivirus, vB_PaeL_PcyII-10_LeviOr01, from hospital waste water. Its genome comprises 3669 nucleotides and encodes four putative proteins. Following bacterial infection, a carrier state is established in a fraction of the cells, conferring superinfection immunity. Such cells also resist other phages that use type IV pili as a receptor. The carrier population is composed of a mixture of cells producing phage, and susceptible cells that are non-carriers. Carrier cells accumulate phage until they burst, releasing large quantities of virions. The continuous presence of phage favours the emergence of host variants bearing mutations in genes involved in type IV pilus biogenesis, but also in genes affecting lipopolysaccharide (LPS) synthesis. The establishment of a carrier state in which phage particles are continuously released was previously reported for some dsRNA phages, but has not previously been described for a levivirus. The present results highlight the importance of the carrier state, an association that benefits both phages and bacteria and plays a role in bacterial evolution.
    Mots-clés : Genome, Viral, Host-Parasite Interactions, Levivirus, LGBMB, MICROBIO, Pseudomonas aeruginosa, Pseudomonas Phages, RNA, Viral, Sequence Analysis, DNA, Virus Release, Virus Replication.

  • V. Timofeev, I. Bakhteeva, G. Titareva, P. Kopylov, D. Christiany, A. Mokrievich, I. Dyatlov, et G. Vergnaud, « Russian isolates enlarge the known geographic diversity of Francisella tularensis subsp. mediasiatica », PloS One, vol. 12, nᵒ 9, p. e0183714, 2017.
    Résumé : Francisella tularensis, a small Gram-negative bacterium, is capable of infecting a wide range of animals, including humans, and causes a plague-like disease called tularemia-a highly contagious disease with a high mortality rate. Because of these characteristics, F. tularensis is considered a potential agent of biological terrorism. Currently, F. tularensis is divided into four subspecies, which differ in their virulence and geographic distribution. Two of them, subsp. tularensis (primarily found in North America) and subsp. holarctica (widespread across the Northern Hemisphere), are responsible for tularemia in humans. Subsp. novicida is almost avirulent in humans. The fourth subspecies, subsp. mediasiatica, is the least studied because of its limited distribution and impact in human health. It is found only in sparsely populated regions of Central Asia. In this report, we describe the first focus of naturally circulating F. tularensis subsp. mediasiatica in Russia. We isolated and characterized 18 strains of this subspecies in the Altai region. All strains were highly virulent in mice. The virulence of subsp. mediasiatica in a vaccinated mouse model is intermediate between that of subsp. tularensis and subsp. holarctica. Based on a multiple-locus variable number tandem repeat analysis (MLVA), we show that the Altaic population of F. tularensis subsp. mediasiatica is genetically distinct from the classical Central Asian population, and probably is endemic to Southern Siberia. We propose to subdivide the mediasiatica subspecies into three phylogeographic groups, M.I, M.II and M.III.
    Mots-clés : Alleles, Animals, Biodiversity, Citrulline, Cluster Analysis, Female, Francisella tularensis, Genotype, Geography, Glycerol, Humans, LGBMB, Male, Mice, Mice, Inbred BALB C, MICROBIO, Minisatellite Repeats, Phylogeography, Polymorphism, Single Nucleotide, Russia, Stem Cells, Tularemia, Vaccination, Virulence.


  • E. C. Hollenbeck, C. Douarche, J. - M. Allain, P. Roger, C. Regeard, L. Cegelski, G. G. Fuller, et E. Raspaud, « Mechanical Behavior of a Bacillus subtilis Pellicle », The Journal of Physical Chemistry. B, vol. 120, nᵒ 26, p. 6080-6088, juill. 2016.
    Résumé : Bacterial biofilms consist of a complex network of biopolymers embedded with microorganisms, and together these components form a physically robust structure that enables bacteria to grow in a protected environment. This structure can help unwanted biofilms persist in situations ranging from chronic infection to the biofouling of industrial equipment, but under certain circumstances it can allow the biofilm to disperse and colonize new niches. Mechanical properties are therefore a key aspect of biofilm life. In light of the recently discovered growth-induced compressive stress present within a biofilm, we studied the mechanical behavior of Bacillus subtilis pellicles, or biofilms at the air-liquid interface, and tracked simultaneously the force response and macroscopic structural changes during elongational deformations. We observed that pellicles behaved viscoelastically in response to small deformations, such that the growth-induced compressive stress was still present, and viscoplastically at large deformations, when the pellicles were under tension. In addition, by using particle imaging velocimetry we found that the pellicle deformations were nonaffine, indicating heterogeneous mechanical properties with the pellicle being more pliable near attachment surfaces. Overall, our results indicate that we must consider not only the viscoelastic but also the viscoplastic and mechanically heterogeneous nature of these structures to understand biofilm dispersal and removal.
    Mots-clés : LGBMB, MICROBIO.

  • M. Imperi, V. Pittiglio, G. D'Avenio, G. Gherardi, A. Ciammaruconi, F. Lista, C. Pourcel, L. Baldassarri, et R. Creti, « A new genotyping scheme based on MLVA for inter-laboratory surveillance of Streptococcus pyogenes », Journal of Microbiological Methods, vol. 127, p. 176-181, août 2016.
    Résumé : A newly developed MLVA seven-loci scheme for Streptococcus pyogenes is described. The method can be successfully applied by using both agarose gel with visual inspections of bands and Lab on Chip technology. The potential of the present MLVA has been tested on a collection of 100 clinical GAS strains representing the most common emm types found in high-income countries plus 18 published gap-free genomes, in comparison to PFGE and MLST. The MLVA analysis defined 30 MLVA types with ten out of the considered 15 emm types exhibiting multiple and specific MLVA types. In only one occasion the same MLVA profile was shared between isolates belonging to two different emm types. A robust congruency between the methods was observed, with MLVA discriminating within clonal complexes as defined by PFGE or MLST. This new MLVA scheme can be adopted as a quick, low-cost and reliable typing method to track the short-term diffusion of GAS clones in inter-laboratory-based surveillance.
    Mots-clés : emm typing, Group A streptococcus, LGBMB, MICROBIO, MLST, MLVA, PFGE, S. pyogenes.

  • L. Latino, C. Midoux, Y. Hauck, G. Vergnaud, et C. Pourcel, « Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa », Microbiology (Reading, England), vol. 162, nᵒ 5, p. 748-763, mai 2016.
    Résumé : Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10- 5 for single phage infection and 10- 6 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure.
    Mots-clés : Alginates, Bacteriophages, Base Sequence, Biofilms, DNA, Bacterial, DNA, Viral, Fimbriae, Bacterial, Genome, Bacterial, Genome, Viral, Glucuronic Acid, Hexuronic Acids, LGBMB, Lipopolysaccharides, Lysogeny, MICROBIO, Mutation, O Antigens, Pseudomonas aeruginosa, Sequence Analysis, DNA.

  • J. R. Osman, A. de Zelicourt, T. Bisseling, R. Geurts, H. Hirt, et M. S. DuBow, « Bacterial Rhizosphere Biodiversity from Several Pioneer Desert Sand Plants Near Jizan, Saudi Arabia », The Open Conference Proceedings Journal, vol. 7, nᵒ suppl 1: M7, p. 70-79, avr. 2016.

  • C. Pourcel, C. Midoux, M. Bourkaltseva, E. Pleteneva, et V. Krylov, « Complete Genome Sequence of PM105, a New Pseudomonas aeruginosa B3-Like Transposable Phage », Genome Announcements, vol. 4, nᵒ 2, mars 2016.
    Résumé : The complete genome of the Pseudomonas aeruginosa bacteriophage PM105 is 39,593 bp long. The phage belongs to the B3 family of transposable Mu-like phages, as confirmed by the presence of bacterial DNA joined to the phage genome ends. PM105, together with other B3-like phages, form a newly arising species.
    Mots-clés : LGBMB, MICROBIO.

  • C. Pourcel, C. Midoux, L. Latino, M. - A. Petit, et G. Vergnaud, « Complete Genome Sequences of Pseudomonas aeruginosa Phages vB_PaeP_PcyII-10_P3P1 and vB_PaeM_PcyII-10_PII10A », Genome Announcements, vol. 4, nᵒ 6, nov. 2016.
    Résumé : vB_PaeP_PcyII-10_P3P1 and vB_PaeM_PcyII-10_PII10A are Pseudomonas aeruginosa bacteriophages belonging, respectively, to the Lit1virus genus of the Podoviridae family and the Pbunavirus genus of the Myoviridae family. Their genomes are 72,778 bp and 65,712 bp long, containing 94 and 93 predicted open reading frames, respectively.
    Mots-clés : LGBMB, MICROBIO.

  • H. C. Scholz, S. Revilla-Fernández, S. Al Dahouk, J. A. Hammerl, M. S. Zygmunt, A. Cloeckaert, M. Koylass, A. M. Whatmore, J. Blom, G. Vergnaud, A. Witte, K. Aistleitner, et E. Hofer, « Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes) », International Journal of Systematic and Evolutionary Microbiology, vol. 66, nᵒ 5, p. 2090-2098, mai 2016.
    Résumé : Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).
    Mots-clés : LGBMB, MICROBIO.

  • G. Vergnaud, G. Girault, S. Thierry, C. Pourcel, N. Madani, et Y. Blouin, « Comparison of French and Worldwide Bacillus anthracis Strains Favors a Recent, Post-Columbian Origin of the Predominant North-American Clade », PloS One, vol. 11, nᵒ 2, p. e0146216, 2016.
    Résumé : BACKGROUND: Bacillus anthracis, the highly dangerous zoonotic bacterial pathogen species is currently composed of three genetic groups, called A, B and C. Group A is represented worldwide whereas group B is present essentially in Western Europe and Southern Africa. Only three strains from group C have been reported. This knowledge is derived from the genotyping of more than 2000 strains collected worldwide. Strains from both group A and group B are present in France. Previous investigations showed that the majority of sporadic French strains belong to the so-called A.Br.011/009 group A clade and define a very remarkable polytomy with six branches. Here we explore the significance of this polytomy by comparing the French B. anthracis lineages to worldwide lineages. We take advantage of whole genome sequence data previously determined for 122 French strains and 45 strains of various origins. RESULTS: A total of 6690 SNPs was identified among the available dataset and used to draw the phylogeny. The phylogeny of the French B group strains which belongs to B.Br.CNEVA indicates an expansion from the south-east part of France (the Alps) towards the south-west (Massif-Central and Pyrenees). The relatively small group A strains belonging to A.Br.001/002 results from at least two independent introductions. Strikingly, the data clearly demonstrates that the currently predominant B. anthracis lineage in North America, called WNA for Western North American, is derived from one branch of the A.Br.011/009 polytomy predominant in France. CONCLUSIONS/SIGNIFICANCE: The present work extends the range of observed substitution rate heterogeneity within B. anthracis, in agreement with its ecology and in contrast with some other pathogens. The population structure of the six branches A.Br.011/009 polytomy identified in France, diversity of branch length, and comparison with the WNA lineage, suggests that WNA is of post-Columbian and west European origin, with France as a likely source. Furthermore, it is tempting to speculate that the polytomy's most recent common ancestor -MRCA- dates back to the Hundred Years' war between France and England started in the mid-fourteenth century. These events were associated in France with deadly epidemics and major economic and social changes.
    Mots-clés : Bacillus anthracis, England, France, Genome analysis, Genome evolution, Genotype, LGBMB, MICROBIO, Molecular genetics, North America, Phylogenetic analysis, Polymorphism, Single Nucleotide, Species diversity, Vaccines.


  • S. An, H. H. Sin, et M. S. DuBow, « Modification of atmospheric sand-associated bacterial communities during Asian sandstorms in China and South Korea », Heredity, vol. 114, nᵒ 5, p. 460-467, 2015.
    Résumé : The transport of desert soil into the atmosphere during desert sandstorms can affect the Earth's climate and environmental health. Asian desert sandstorms occur almost every year during the Spring, as the atmosphere in the Northern hemisphere warms. It is conceivable that these Asian desert sandstorms may transport microbes from deserts, such as the Gobi and Taklamaken deserts, over long distances in China, east Asia and the Pacific. In this study, we examined local atmospheric sand particle-associated bacterial populations collected in the absence (sterile sand exposed for 24 h to the air in the absence of a sandstorm) and presence of sandstorms in five Asian cities. We used pyrosequencing of PCR-amplified 16S rDNA genes from sand-extracted total DNA to overcome cultivation limitations of bacterial enumeration. We found that >90% of the control and sandstorm sequences could be classified as representing bacteria belonging to four phyla: Proteobacteria, Bacteriodetes, Actinobacteria and Firmicutes. The sand-associated bacterial populations in sandstorm samples were distinct from sand-associated bacteria in the absence of a sandstorm. Members of the phylum Proteobacteria were found to significantly increase in sandstorm samples (P=0.01). Principal component analyses showed that the sand-associated bacterial populations were best clustered by sampling year, rather than location. DNA sequences representing bacteria belonging to several genera (including putative human pathogens) were observed to increase in sand-associated samples from sandstorms, whereas others were found to decrease, when comparing sand-associated bacterial populations versus those in control samples, suggesting human/environmental implications of sandstorm events.
    Mots-clés : Air Microbiology, Bacteria, Bayes Theorem, China, DNA, Bacterial, Environmental Monitoring, LGBMB, MICROBIO, Republic of Korea, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Silicon Dioxide, Wind.

  • S. Ciarroni, L. Gallipoli, M. C. Taratufolo, M. I. Butler, R. T. M. Poulter, C. Pourcel, G. Vergnaud, G. M. Balestra, et A. Mazzaglia, « Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide », PloS One, vol. 10, nᵒ 8, p. e0135310, 2015.
    Résumé : The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples.
    Mots-clés : Actinidia, Genetic Loci, Genome, Bacterial, Haplotypes, High-Throughput Nucleotide Sequencing, LGBMB, MICROBIO, Minisatellite Repeats, Multilocus Sequence Typing, Phylogeny, Plant Diseases, Polymorphism, Single Nucleotide, Pseudomonas syringae.

  • C. Essoh, L. Latino, C. Midoux, Y. Blouin, G. Loukou, S. - P. A. Nguetta, S. Lathro, A. Cablanmian, A. K. Kouassi, G. Vergnaud, et C. Pourcel, « Investigation of a Large Collection of Pseudomonas aeruginosa Bacteriophages Collected from a Single Environmental Source in Abidjan, Côte d'Ivoire », PloS One, vol. 10, nᵒ 6, p. e0130548, 2015.
    Résumé : Twenty two distinct bacteriophages were isolated from sewage water from five locations in the city of Abidjan, Côte d'Ivoire over a two-year period, using a collection of Pseudomonas aeruginosa strains with diverse genotypes. The phages were characterized by their virulence spectrum on a panel of selected P. aeruginosa strains from cystic fibrosis patients and by whole genome sequencing. Twelve virions representing the observed diversity were visualised by electron microscopy. The combined observations showed that 17 phages, distributed into seven genera, were virulent, and that five phages were related to temperate phages belonging to three genera. Some showed similarity with known phages only at the protein level. The vast majority of the genetic variations among virulent phages from the same genus resulted from seemingly non-random horizontal transfer events, inside a population of P. aeruginosa phages with limited diversity. This suggests the existence of a single environmental reservoir or ecotype in which continuous selection is taking place. In contrast, mostly point mutations were observed among phages potentially capable of lysogenisation. This is the first study of P. aeruginosa phage diversity in an African city and it shows that a large variety of phage species can be recovered in a limited geographical site at least when different bacterial strains are used. The relative temporal and spatial stability of the Abidjan phage population might reflect equilibrium in the microbial community from which they are released.
    Mots-clés : Bacteriophages, Cote d'Ivoire, Genome, Viral, LGBMB, MICROBIO, Microscopy, Electron, Molecular Sequence Data, Pseudomonas aeruginosa, Virion.

  • Y. Hauck, C. Soler, P. Gérôme, R. Vong, C. Macnab, G. Appere, G. Vergnaud, et C. Pourcel, « A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes », Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, vol. 33, p. 233-241, juill. 2015.
    Résumé : Propionibacterium acnes plays a central role in the pathogenesis of acne and is responsible for severe opportunistic infections. Numerous typing schemes have been developed that allow the identification of phylotypes, but they are often insufficient to differentiate subtypes. To better understand the genetic diversity of this species and to perform epidemiological analyses, high throughput discriminant genotyping techniques are needed. Here we describe the development of a multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) method. Thirteen VNTRs were identified in the genome of P. acnes and were used to genotype a collection of clinical isolates. In addition, publically available sequencing data for 102 genomes were analyzed in silico, providing an MLVA genotype. The clustering of MLVA data was in perfect congruence with whole genome based clustering. Analysis of the clustered regularly interspaced short palindromic repeat (CRISPR) element uncovered new spacers, a supplementary source of genotypic information. The present MLVA13 scheme and associated internet database represents a first line genotyping assay to investigate large number of isolates. Particular strains may then be submitted to full genome sequencing in order to better analyze their pathogenic potential.
    Mots-clés : Cluster Analysis, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR, Genes, Bacterial, Genotyping, Gram-Positive Bacterial Infections, Humans, In silico MLVA, LGBMB, MICROBIO, Minisatellite Repeats, Molecular Typing, Multilocus Sequence Typing, Post-surgery infection, Propionibacterium acnes, Sequence Analysis, DNA, VNTR.

  • K. A. Koskela, L. Mattinen, L. Kalin-Mänttäri, G. Vergnaud, O. Gorgé, S. Nikkari, et M. Skurnik, « Generation of a CRISPR database for Yersinia pseudotuberculosis complex and role of CRISPR-based immunity in conjugation », Environmental Microbiology, vol. 17, nᵒ 11, p. 4306-4321, nov. 2015.
    Résumé : The clustered regularly interspaced short palindromic repeat - CRISPR-associated genes (CRISPR-Cas) system is used by bacteria and archaea against invading conjugative plasmids or bacteriophages. Central to this immunity system are genomic CRISPR loci that contain fragments of invading DNA. These are maintained as spacers in the CRISPR loci between direct repeats and the spacer composition in any bacterium reflects its evolutionary history. We analysed the CRISPR locus sequences of 335 Yersinia pseudotuberculosis complex strains. Altogether 1902 different spacer sequences were identified and these were used to generate a database for the spacer sequences. Only ∼10% of the spacer sequences found matching sequences. In addition, surprisingly few spacers were shared by Yersinia pestis and Y. pseudotuberculosis strains. Interestingly, 32 different protospacers were present in the conjugative plasmid pYptb32953. The corresponding spacers were identified from 35 different Y. pseudotuberculosis strains indicating that these strains had encountered pYptb32953 earlier. In conjugation experiments, pYptb32953-specific spacers generally prevented conjugation with spacer-positive and spacer-free strains. However, some strains with one to four spacers were invaded by pYptb32953 and some spacer-free strains were fully resistant. Also some spacer-positive strains were intermediate resistant to conjugation. This suggests that one or more other defence systems are determining conjugation efficiency independent of the CRISPR-Cas system.
    Mots-clés : Bacteriophages, Base Sequence, Clustered Regularly Interspaced Short Palindromic Repeats, Conjugation, Genetic, Databases, Nucleic Acid, Genomics, LGBMB, MICROBIO, Molecular Sequence Data, Plasmids, Yersinia pestis, Yersinia pseudotuberculosis.

  • Z. Kovalova, M. Leroy, C. Jacobs, M. J. Kirkpatrick, Z. Machala, F. Lopes, C. O. Laux, M. S. DuBow, et E. Odic, « Atmospheric pressure argon surface discharges propagated in long tubes: physical characterization and application to bio-decontamination », Journal of Physics D: Applied Physics, vol. 48, nᵒ 46, p. 464003, nov. 2015.

  • A. Pinhas, M. Dubow, N. Shah, E. Cheang, C. L. Liu, M. Razeen, A. Gan, R. Weitz, Y. N. Sulai, T. Y. Chui, A. Dubra, et R. B. Rosen, « FELLOW EYE CHANGES IN PATIENTS WITH NONISCHEMIC CENTRAL RETINAL VEIN OCCLUSION: Assessment of Perfused Foveal Microvascular Density and Identification of Nonperfused Capillaries », Retina (Philadelphia, Pa.), vol. 35, nᵒ 10, p. 2028-2036, oct. 2015.
    Résumé : PURPOSE: Eyes fellow to nonischemic central retinal vein occlusion (CRVO) were examined for abnormalities, which might explain their increased risk for future occlusion, using adaptive optics scanning light ophthalmoscope fluorescein angiography. METHODS: Adaptive optics scanning light ophthalmoscope fluorescein angiography foveal microvascular densities were calculated. Nonperfused capillaries adjacent to the foveal avascular zone were identified. Spectral domain optical coherence tomography, ultrawide field fluorescein angiographies, and microperimetry were also performed. RESULTS: Ten fellow eyes of nine nonischemic CRVO and 1 nonischemic hemi-CRVO subjects and four affected eyes of three nonischemic CRVO and one nonischemic hemi-CRVO subjects were imaged. Ninety percent of fellow eyes and 100% of affected eyes demonstrated at least 1 nonperfused capillary compared with 31% of healthy eyes. Fellow eye microvascular density (35 ± 3.6 mm(-1)) was significantly higher than that of affected eyes (25 ± 5.2 mm(-1)) and significantly lower than that of healthy eyes (42 ± 4.2 mm(-1)). Compared with healthy controls, spectral domain optical coherence tomography thicknesses showed no significant difference, whereas microperimetry and 2/9 ultrawide field fluorescein angiography revealed abnormalities in fellow eyes. CONCLUSION: Fellow eye changes detectable on adaptive optics scanning light ophthalmoscope fluorescein angiography reflect subclinical pathology difficult to detect using conventional imaging technologies. These changes may help elucidate the pathogenesis of nonischemic CRVO and help identify eyes at increased risk of future occlusion.
    Mots-clés : Adult, Aged, Capillaries, Female, Fluorescein Angiography, Functional Laterality, Humans, Ischemia, LGBMB, Male, MICROBIO, Middle Aged, Regional Blood Flow, Retinal Vein Occlusion, Retinal Vessels, Retrospective Studies, Tomography, Optical Coherence, Visual Field Tests, Visual Fields, Young Adult.

  • C. Segonds, M. Thouverez, A. Barthe, N. Bossuet-Greif, L. Tisseyre, P. Plésiat, G. Vergnaud, G. Chabanon, et C. Pourcel, « Development of a multiple-locus variable-number tandem-repeat typing scheme for genetic fingerprinting of Burkholderia cenocepacia and application to nationwide epidemiological analysis », Journal of Clinical Microbiology, vol. 53, nᵒ 2, p. 398-409, févr. 2015.
    Résumé : Organisms of the Burkholderia cepacia complex are especially important pathogens in cystic fibrosis (CF), with a propensity for patient-to-patient spread and long-term respiratory colonization. B. cenocepacia and Burkholderia multivorans account for the majority of infections in CF, and major epidemic clones have been recognized throughout the world. The aim of the present study was to develop and evaluate a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for B. cenocepacia. Potential VNTR loci were identified upon analysis of the annotated genome sequences of B. cenocepacia strains AU1054, J2315, and MCO-3, and 10 of them were selected on the basis of polymorphisms and size. A collection of 100 B. cenocepacia strains, including epidemiologically related and unrelated strains, as well as representatives of the major epidemic lineages, was used to evaluate typeability, epidemiological concordance, and the discriminatory power of MLVA-10 compared with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Longitudinal stability was assessed by testing 39 successive isolates from 14 patients. Typeability ranged from 0.91 to 1, except for that of one marker, which was not amplified in 53% of the B. cenocepacia IIIA strains. The MLVA types were shown to be stable in chronically colonized patients and within outbreak-related strains, with excellent epidemiological concordance. Epidemic and/or globally distributed lineages (epidemic Edinburgh-Toronto electrophoretic type 12 [ET-12], sequence type 32 [ST-32], ST-122, ST-234, and ST-241) were successfully identified. Conversely, the discriminatory power of MLVA was lower than that of PFGE or MLST, although PFGE variations within the epidemic lineages sometimes masked their genetic relatedness. In conclusion, MLVA represents a promising cost-effective first-line tool in B. cenocepacia surveillance.
    Mots-clés : Burkholderia cepacia, Burkholderia Infections, Cluster Analysis, Computational Biology, Cystic Fibrosis, DNA Fingerprinting, Genetic Variation, Genome, Bacterial, Genotype, Humans, LGBMB, MICROBIO, Minisatellite Repeats, Molecular Epidemiology, Molecular Typing.

  • A. Touati, Y. Blouin, P. Sirand-Pugnet, H. Renaudin, T. Oishi, G. Vergnaud, C. Bébéar, et S. Pereyre, « Molecular Epidemiology of Mycoplasma pneumoniae: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology », Journal of Clinical Microbiology, vol. 53, nᵒ 10, p. 3182-3194, oct. 2015.
    Résumé : Molecular typing of Mycoplasma pneumoniae is an important tool for identifying grouped cases and investigating outbreaks. In the present study, we developed a new genotyping method based on single nucleotide polymorphisms (SNPs) selected from the whole-genome sequencing of eight M. pneumoniae strains, using the SNaPshot minisequencing assay. Eight SNPs, localized in housekeeping genes, predicted lipoproteins, and adhesin P1 genes were selected for genotyping. These SNPs were evaluated on 140 M. pneumoniae clinical isolates previously genotyped by multilocus variable-number tandem-repeat analysis (MLVA-5) and adhesin P1 typing. This method was also adapted for direct use with clinical samples and evaluated on 51 clinical specimens. The analysis of the clinical isolates using the SNP typing method showed nine distinct SNP types with a Hunter and Gaston diversity index (HGDI) of 0.836, which is higher than the HGDI of 0.583 retrieved for the MLVA-4 typing method, where the nonstable Mpn1 marker was removed. A strong correlation with the P1 adhesin gene typing results was observed. The congruence was poor between MLVA-5 and SNP typing, indicating distinct genotyping schemes. Combining the results increased the discriminatory power. This new typing method based on SNPs and the SNaPshot technology is a method for rapid M. pneumoniae typing directly from clinical specimens, which does not require any sequencing step. This method is based on stable markers and provides information distinct from but complementary to MLVA typing. The combined use of SNPs and MLVA typing provides powerful discrimination of strains.
    Mots-clés : Adhesins, Bacterial, Adolescent, Child, Child, Preschool, Female, Genes, Essential, Genotyping Techniques, Humans, Infant, LGBMB, Male, MICROBIO, Molecular Epidemiology, Molecular Typing, Mycoplasma pneumoniae, Pneumonia, Mycoplasma, Polymorphism, Single Nucleotide.
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Publications avant 2015

- Winter C, Garcia JA, Weinbauer MG, DuBow MS, Herndl GJ. Comparison of Deep-Water Viromes from the Atlantic Ocean and the Mediterranean Sea. PloS One. 9 : e100600. (2014).

- Jakubek D, Guillaume C, Binet, M, Leblon G, DuBow MS and Le Brun M. Susceptibility of Legionella strains to the chlorinated biocide, Monochloramine. Microbes and Environments 28 : 336-345 (2013).

- An S, Couteau C, Luo F, Neveu J and DuBow MS. Bacterial diversity of surface sand samples from the Gobi and Taklamaken deserts. Microbial Ecology. 66 : 850-860 (2013).

- Jakubek D, Le Brun M, Leblon G, DuBow MS and Binet, M. The impact of monochloramine on the diversity and dynamics of Legionella pneumophila subpopulations in a nuclear power plant cooling circuit. FEMS Microbiology Ecology. 85 : 302-312 (2013).

- Prestel E, Regeard C, Salamitou S, Neveu, J and DuBow MS. The bacteria and bacteriophages from a Mesquite Flats site of the Death Valley desert. Antonie van Leeuwenhoek. 103 : 1329-1341 (2013).

- Bricheux B, Morin L, Le Moal G, Coffe G, Balestrino D, Charbonnel N, Bohatier J and Forestier C. Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river. Microbiology Open. 2 : 402-414 (2013)

- Trejoa M, Douarchea C, Bailleuxa V, Poularda C, Mariota S, Regeard C and Raspauda E. Elasticity and wrinkled morphology of Bacillus subtilis pellicles. Proceedings of the National Academy of Sciences (USA). 110 : 2011-2016 (2013).

- Jakubek D, Le Brun M, Leblon G, DuBow MS and Binet, M. Validation of IRS PCR, a molecular typing method, for the study of the diversity and population dynamics of Legionella in industrial cooling circuits. Letters in Applied Microbiology. 56 : 135-141 (2013).

- Sauge-Merle S, Lecomte-Pradines C, Carrier P, Cuine S and DuBow MS. Heavy metal accumulation by recombinant mammalian metallothionein within Escherichia coli protects against elevated metal exposure. Chemosphere. 88 : 918–924 (2012).

- Ray JL, Töpper B, An S, Silyakova A, Spindelböck J, Thyrhaug R, DuBow MS, Thingstad TF and Sandaa R-A. Effect of increased pCO2 on bacterial assemblage shifts in response to glucose addition in Fram Strait seawater mesocosms. FEMS Microbiology Ecology. 82:713-723 (2012).

- Prestel E, Regeard C, Andrews J, Oger P and DuBow MS. A novel bacteriophage morphotype with a ribbon-like structure at the tail extremity. Research Journal of Microbiology. 7 : 75-81 (2012).

- Mezanges X, Regeard C, Gerin C, Deroulers C, Grammaticos B and Badoual M. Modeling the role of water in Bacillus subtilis colonies. Physical Review E 85:04193 (2012).

- Cirou A, Mondy S, An S, Charrier A, Sarrazin A, Thoison O, DuBow MS and Faure D. Efficient biostimulation of the native and introduced quorum-quenching Rhodococcus erythropolis is revealed by a combination of analytical chemistry, microbiology and pyrosequencing. Applied and Environmental Microbiology. 78 : 481-492 (2012).

- Poitelon J-B, Joyeux M, Welté B, Duguet J-P, and DuBow MS. The drinking water distribution network : a complex ecosystem related to public health issues. Journal of Water Science. 24 : 383-418 (2011).

- De Luca G, Barakat M, Ortet P, Fochesato S, Jourlin-Castelli C, Ansaldi M, Py B, Fichant G, Coutinho PM, Voulhoux R, Bastien O, Maréchal E, Henrissat B, Quentin Y, Noirot PH, Filloux A, Méjean V, DuBow MS, Barras F, Barbe V, Weissenbach J, Mihalcescu I, Verméglio A, Achouak W and Heulin T. The cyst-dividing bacterium Ramlibacter tataouinensis TTB310 genome reveals a well-stocked toolbox for adaptation to a desert environment. PloS One. 6 : e23784. (2011).

- Maillard J, Charnay M-P, Regeard C, Rohrbach-Brandt E, Rouzeau-Szynalski K, Rossi P and Holliger C. Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases. Bodegradation. 22 : 949-960 (2011).

- Neveu J, Regeard C and DuBow MS. Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts. Applied Microbiology and Biotechnology. 91 : 635-644 (2011).

- Marchal M, Briandet R, Halter D, Koechler S, DuBow MS, Lett M-C and Bertin PM. Subinhibitory arsenite concentrations lead to population dispersal in Thiomonas sp. PloS One. 6 : e23181 (2011).

- Deschavanne P, DuBow MS and Regeard C. The use of genomic signature distance between bacteriophages and their hosts displays evolutionary relationships and phage growth cycle determination. Virology Journal 7:163 (2010).

- Poitelon J-B, Joyeux M, Welté B, Duguet J-P, Prestel E and DuBow MS. Variations of bacterial 16S rDNA phylotypes prior to and after chlorination for drinking water production from two surface water treatment plants. Journal of Industrial Microbiology and Biotechnology. 37 : 117-128 (2010).

- Salamitou S, Kirkpatrick MJ, Ly HM, Leblon G, Odic E and DuBow MS. Augmented Survival of Bacteria Within Biofilms to Exposure to an Atmospheric Pressure Non-Thermal Plasma Source. Biotechnology. 8 : 228-234 (2009).

- Dehbi M, Moeck G, Arhin FF, Bauda P, Bergeron D, Kwan T, Liu J, McCarty J, DuBow MS and Pelletier J. Inhibition of Transcription in Staphylococcus aureus by a Primary Sigma Factor-Binding Polypeptide from Phage G1. Journal of Bacteriology. 191 : 3763–3771 (2009).

- Poitelon J-B, Joyeux M, Welté B, Duguet J-P, Peplies J and DuBow MS. Identification and phylogeny of eukaryotic 18S rDNA phylotypes detected in chlorinated finished drinking water samples from three Parisian surface water treatment plants. Letters in Applied Microbiology. 49 : 589 – 595 (2009).

- Poitelon J-B, Joyeux M, Welté B, Duguet J-P, Prestel E, Lespinet O and DuBow MS. Assessment of phylogenetic diversity of bacterial microflora in drinking water using serial analysis of ribosomal sequence tags. Water Research. 43 : 4197-4206 (2009).

- Prestel E, Salamitou, S and DuBow MS. An examination of the bacteriophages and bacteria of the Namib Desert. Journal of Microbiology. 46 : 364-372 (2008).

- Leroy M, Prigent M, Dutertre M, Confalonieri F and DuBow MS. Bacteriophage morphotype and genome diversity in Seine River sediment. Freshwater Biology. 53:1176-1185 (2008).

- Gueuné H, Durand MJ, Thouand G and DuBow MS. The ygaVP genes of Escherichia coli form a tributyltin-inducible operon. Applied and Environmental Microbiology. 74:1954-1958 (2008).

- Phrommavanh V, Klein J, Descostes M, Beaucaire C, Gaudet JP, Prestel E, DuBow MS, and Laporte E. Role of bacteria on uranium migration in a calcareous peatland. Geochimica and Cosmochimica Acta. 71 : A788 (2007).

- Belley A, Callejo M, Arhin F, Dehbi M, Fadhil I, Liu J, McKay G, Srikumar R, Bauda P, Ha N, DuBow MS, Gros P, Pelletier J, and Moeck G. Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus. Molecular Microbiology. 62 : 1132-1143 (2006).

- Kwan T, Liu J, DuBow MS, Gros P and Pelletier J. Comparative genomic analysis of 18 Pseudomonas aeruginosa bacteriophages. Journal of Bacteriology. 188 : 1184-1187 (2006).

- Durand MJ, DuBow MS, Horry H, Picart P, Daniel P, S Lebeau and Thouand G. Detection of tributyl and dibutyl tin in the environment with a bioluminescent bacteria : from the bioassay to the biosensor. Trends in Agriculture and Soil Pollution Research. 1 : 1-42 (2006).

- Prigent M, Leroy M, Confalonieri F, Dutertre M and DuBow MS. A diversity of bacteriophage forms and genomes can be isolated from the surface sands of the Sahara Desert. Extremophiles. 9 : 289-296 (2005).

- Kwan T, Liu J, DuBow MS, Gros P and Pelletier J. The complete annotated genome sequences of 27 Staphylococcus aureus bacteriophages. Proc. Natl. Acad. Sci. USA. 102 : 5174-5179 (2005).

- Liu J, Dehbi M, Moeck G, Arhin F, Bauda P, Bergeron D, Callejo M, Ferretti V, Ha N, Kwan T, McCarty J, Srikumar R, Williams D, Wu JJ, Gros P, Pelletier J, and DuBow MS. Antimicrobial drug discovery through bacteriophage genomics. Nature Biotechnology. 22:185-191 (2004).

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