Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

Diversity of Vibrionaceae bacteria isolated from the haemolymph of the spider crab Maja brachydactyla

Aims: The aim of this study was to characterise and identify vibrios isolated from the haemolymph of apparently healthy adult spider crabs (Maja brachydactyla) wild-caught in the Spanish localities of Galician coast and in the Canary Islands and also from captive animals held at IRTA’s facilities in the Ebro Delta of Catalonia, north-west Spanish Mediterranean coast. Methods and Results: A total of 277 bacterial isolates were obtained, and of these, 171 were characterised with rep-PCR, resulting electrophoretic bands were analysed and clusters formed. Identification of representative strains of each cluster was made by sequencing the 16S rRNA. Samples from animals caught in Galicia and captive at IRTA (around 15–18 C) rendered mostly species belonging to the Splendidus clade (72Æ2 and 76Æ6% respectively), commonly found in cold waters (below 20 C). Higher species diversity was found in the haemolymph of the captive animals. In the warmer Canary Islands waters (around 21 C), the diversity of vibrios is dominated by three clades, Harveyi (Vibrio core group, 39Æ3%), Orientalis (23Æ2%) and Splendidus (21Æ4%) with a species diversity that equals that of the colder captive animals. Conclusions: Differences in the vibrios populations were found in the haemolymph extracted from animals collected from the three localities. Potential new species were found, and their description is under way. Significance and Impact of Study: As with other invertebrates, spider crabs also contain a diverse population of vibrios. These findings should help researchers to diagnose when a crab is infected.

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells.

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer's patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c(+)CD11b(+)CD8(-) dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

Intestinal bacteria condition dendritic cells to promote IgA production.

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyer's Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-beta, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.

Lower faecal egg excretion in chemically-induced diabetic mice infected with Schistosoma mansoni due to impaired egg maturation

The effect of streptozotocin-induced diabetes mellitus was studied in mice infected with Schistosoma mansoni. Faecal egg excretion was lower in diabetic mice but worm load and total amount of eggs in the intestine tissue were equal to the control group. Evaluation of an oogram showed a great number of immature dead eggs and a low number of mature eggs in diabetic mice. It was therefore concluded that faecal egg excretion was lower in diabetic mice due to impaired egg maturation.

Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction.

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth. STUDY DESIGN: Amniotic fluid samples (14-18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR. RESULTS: Amniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group (P=0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P=0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group (P=0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR=0.289, 95%CI=0.1-0.9, P=0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups. CONCLUSIONS: High IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.

Comparison of the Proportion Method With Mycobacteria Growth Indicator Tube and E-test for Susceptibility Testing of Mycobacterium tuberculosis

The aim of this study was to investigate the correlation between proportion method with mycobacteria growth indicator tube (MGIT) and E-test for Mycobacterium tuberculosis. Forty clinical isolates were tested. MGIT and E-test with the first line antituberculous drugs correlated with the proportion method. Our results suggested that MGIT and E-test methods can be routinely used instead of the proportion method.

Enterocytozoon bieneusi (microsporidia) in faecal samples from domestic animals from Galicia, Spain

In this survey we examined 87 domestic animal stool samples in order to detect the possible presence of microsporidia in animals in close contact with humans in Galicia (NW, Spain). The detection of Enterocytozoon bieneusi spores was confirmed in faecal samples from two dogs and one goat by polymerase chain reaction. None of the positive samples for microsporidia in the staining method were amplified with species-specific primers for Encephalitozoon intestinalis, E. hellem and E. cuniculi. Four rabbits faecal samples reacted with anti-E. cuniculi serum. Our results could indicate the importance of domestic animals as zoonotic reservoirs of microsporidial human infections.

Abundància i persistència de Faecalibacteriun prausnitzii en la Malaltia de Crohn

Faecalibacterium prausnitzii és un del bacteris anaeròbis més abundants entre les espècies comensals del tracte intestinal humà sa. Aquesta espècie és una de les principals productores de butirat a l'intestí (que és la principal font d’energia per als colonòcits), però també s'ha suggerit que pot produir compostos antiinflamatoris i intervenir en la regulació de vàries rutes metabòliques de l’hoste. F. prausnitzii és un bacteri difícil de cultivar, ja que presenta una elevada sensibilitat a l'oxigen i presenta uns requeriments nutricionals molt exigents, el que ha compromès considerablement el nombre d’estudis basats en aïllats d’aquesta espècie. No obstant això, en els darrers anys l’interès en aquest bacteri està creixent ja que s’ha evidenciat que les poblacions de F. prausnitzii són variables en diferents grups d'edat i que es veuen reduïdes en certs trastorns intestinals com ara la malaltia inflamatòria intestinal i el càncer colorectal. L’objectiu d'aquest treball ha estat aprofundir en el rol que desenvolupa F. prausnitzii com un dels principals bacteris comensals del tracte intestinal humà. En primer lloc, s’ha dissenyat, optimitzat i validat un nou mètode molecular per determinar l’abundància d’aquesta espècie en mostres del tracte gastrointesinal, i s’ha demostrat la seva possible aplicació per ajudar al diagnòstic de la malaltia de Crohn. En segon lloc, s’ha dut a terme un estudi de les característiques filogenètiques i fenotípiques dels aïllats de F. Prausnitzii disponibles en l'actualitat a fi de coneixre’n millor la diversitat genètica i fenotípica i dilucidar quins factors són crucials en comprometre la població d’aquest bacteri en un intestí malalt. L’anàlisi de les soques ha revelat que F. prausnitzii inclou Principalment dos filogrups, nutricionalment versàtils i molt sensibles a canvis en les condicions ecològiques que pot patir l’intestí de l’hoste sota certes malalties intestinals. En conclusió, els resultat obtinguts en aquest estudi mostren que F. prausnitzii és una espècie ben establerta al còlon sa, amb una elvada versatilitat metabòlica ja que és capaç d’ interactuar amb carbohidrats de diferent estructura i complexitat. S’ha corroborat que aquest microorganisme seria un bon indicador de salut intestinal ja que la seva abundància es veu significativament reduida en pacients amb malaltia de Crohn. Aquests resultats concorden amb els obtinguts per proves fisiològiques que mostren una elevada sensibilitat de l’espècie a determinades condicions relacionades amb malalties intestinals. Estudis futurs s’orientaran a comprendre millor quins factros derrivats de la interacció amb l’hoste també determinen la persistència d’aquesta espècie en un intestí sa o malalt.

Systemic antibody responses to gut commensal bacteria during chronic HIV-1 infection.

BACKGROUND: Human systemic antibody responses to commensal microbiota are not well characterised during health and disease. Of particular interest is the analysis of their potential modulation caused by chronic HIV-1 infection which is associated with sustained enteropathy and systemic B cell disturbances reflected by impaired B cell responses and chronic B cell hyperactivity. The mechanisms underlying B cell hyperactivation and the specificities of the resulting hypergammaglobulinaemia are only poorly understood. METHODS: By a technique referred to as live bacterial FACS (fluorescence-activated cell sorting), the present study investigated systemic antibody responses to several gut and skin commensal bacteria as well as Candida albicans in longitudinal plasma and serum samples from healthy donors, chronic HIV-1-infected individuals with or without diarrhoea and patients with inflammatory bowel disease (IBD). RESULTS: The data show that systemic antibody responses to the commensal microbiota were abundantly present in humans and remained remarkably stable over years. Overall systemic antibody responses to gut commensal bacteria were not affected during chronic HIV-1 infection, with titres decreasing when normalised to elevated plasma immunoglobulin G (IgG) levels found in patients with HIV. In contrast, increases in the titres of high affinity antimicrobiota antibodies were detected in patients with IBD, demonstrating that conditions with known increased intestinal permeability and aberrant mutualism can induce changes in antibody titres observed in these assays. CONCLUSION: Neither HIV-associated enteropathy nor B cell dysfunction impact on the high-affinity systemic antibody responses to gut commensal bacteria. HIV-associated hypergammaglobulinaemia is therefore unlikely to be driven by induction of antimicrobiota antibodies.

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer: reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60ºC, for 30 min, and among them, 12 (18.75%) were resistant at 60ºC, for 60 min. Also, none strain was resistant at 100ºC. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.

Survey of antimicrobial susceptibility patterns of the bacteria of the Bacteroides fragilis group isolated from the intestinal tract of children

The bacteria of the Bacteroides fragilis group are considered important clinical pathogens and they are the most common anaerobes isolated from human endogenous infections. In this study, the susceptibility patterns to antibiotics and metals of 114 species of the B. fragilis group isolated from children with and without diarrhea were determined. Susceptibility was assayed by using an agar dilution method with Wilkins-Chalgren agar. All B. fragilis strains were resistant to lead and nickel, but susceptible to metronidazole and imipenem. beta-lactamase production was detected by using biological and nitrocefin methods, respectively, in 50% and 90.6% of the isolates of children with diarrhea and in 60% and 90% of the isolates of children without diarrhea. Our results show an increase of antibiotics and metals resistance in this microbial group, and a periodic evaluation of the antimicrobial susceptibility is needed. In Brazil, the contamination for antibiotics or metal ions is often observed, and it is suggested an increase the antimicrobial resistance surveillance of this microbial group, mainly those isolated from children's diarrhea.

Application of control measures for infections caused by multi-resistant gram-negative bacteria in intensive care unit patients

Multi-resistant gram-negative rods are important pathogens in intensive care units (ICU), cause high rates of mortality, and need infection control measures to avoid spread to another patients. This study was undertaken prospectively with all of the patients hospitalized at ICU, Anesthesiology of the Hospital São Paulo, using the ICU component of the National Nosocomial Infection Surveillance System (NNIS) methodology, between March 1, 1997 and June 30, 1998. Hospital infections occurring during the first three months after the establishment of prevention and control measures (3/1/97 to 5/31/97) were compared to those of the last three months (3/1/98 to 5/31/98). In this period, 933 NNIS patients were studied, with 139 during the first period and 211 in the second period. The overall rates of infection by multi-resistant microorganisms in the first and second periods were, respectively, urinary tract infection: 3.28/1000 patients/day; 2.5/1000 patients/day; pneumonia: 2.10/1000 patients/day; 5.0/1000 patients/day; bloodstream infection: 1.09/1000 patients/day; 2.5/1000 patients/day. A comparison between overall infection rates of both periods (Wilcoxon test) showed no statistical significance (p = 0.067). The use of intervention measures effectively decreased the hospital bloodstream infection rate (p < 0.001), which shows that control measures in ICU can contribute to preventing hospital infections.