Análise dos aspectos biológicos e epidemiológicos de cepas de Escherichia coli associadas à infecção do trato urinário (ITU)

A invasão de bactérias no trato urinário caracteriza a infecção do sistema urinário. A Escherichia coli é o principal microrganismo associado a esta infecção devido a sua importância em causar ITU, recebeu a denominação de UPEC (Escherichia coli uropatogênica). No presente trabalho pesquisamos em 50 cepas de UPEC, inicialmente isolados de urina de pacientes ambulatoriais com infecções sintomática ou assintomática, a presença de 7 fatores de virulência, através das técnicas de PCR simples e multiplex para verificação dos genes que codificam adesinas P (pap) , fímbria S (sfa), adesina afimbrial (afa), sideroforo (aerobactina- aer), toxinas fator necrotizante citotóxico (cnf) e alfa-hemolisinas (hly), proteína de membrana (traT); ilhas de patogenicidade (virulência) através do marcador PAI. O marcador pCVD432 de EAEC também foi pesquisado nestas amostras. O método difusão em disco foi o utilizado para a determinação dos testes de susceptibilidade aos antimicrobianos. Podemos observar duas faixas etárias de maior incidência de ITU entre as mulheres: 19 a 35 anos, e acima de 50 anos. Sessenta e oito por cento das amostras apresentaram pelo menos um fator de virulência, onde os genes traT (54%) e aer (34%) foram os mais prevalentes. A sequência pCVD432 foi detectado em 6 amostras. No entanto, no ensaio de adesão em células Hep-2, doze amostras não apresentaram aderência (NA 24%). Nas 38 cepas restantes, 24 (48%) apresentaram aderência agregativa (AA). Observamos aderência sem padrão típico (SPT) em 48% das amostras, tendo sido dividido em discreto (SPT-D 22%), moderado (SPT-M 18%) e intenso (SPT-I 8%). Notamos os seguintes perfis de resistência para os antimicrobianos testados: ampicilina (44%), gentamicina (8%), nitrofurantoína (2%), norfloxacino (18%) e sulfametozaxol-trimetoprima (34%).

Photolysis of iron–siderophore chelates promotes bacterial–algal mutualism

Marine microalgae support world fisheries production and influence climate through various mechanisms. They are also responsible for harmful blooms that adversely impact coastal ecosystems and economies. Optimal growth and survival of many bloom-forming microalgae, including climatically important dinoflagellates and coccolithophores, requires the close association of specific bacterial species, but the reasons for these associations are unknown. Here, we report that several clades of Marinobacter ubiquitously found in close association with dinoflagellates and coccolithophores produce an unusual lower-affinity dicitrate siderophore, vibrioferrin (VF). Fe-VF chelates undergo photolysis at rates that are 10–20 times higher than siderophores produced by free-living marine bacteria, and unlike the latter, the VF photoproduct has no measurable affinity for iron. While both an algal-associated bacterium and a representative dinoflagellate partner, Scrippsiella trochoidea, used iron from Fe-VF chelates in the dark, in situ photolysis of the chelates in the presence of attenuated sunlight increased bacterial iron uptake by 70% and algal uptake by >20-fold. These results suggest that the bacteria promote algal assimilation of iron by facilitating photochemical redox cycling of this critical nutrient. Also, binary culture experiments and genomic evidence suggest that the algal cells release organic molecules that are used by the bacteria for growth. Such mutualistic sharing of iron and fixed carbon has important implications toward our understanding of the close beneficial interactions between marine bacteria and phytoplankton, and the effect of these interactions on algal blooms and climate.

鱼腥藻PCC7120受缺铁诱导的外膜蛋白

利用双相法结合蔗糖密度梯度超离心制备获得高纯度的鱼腥藻PCC7120(Anabaena sp.PCC7120)外膜,以双向凝胶电泳分析受缺铁诱导的外膜蛋白。发现缺铁导致5种外膜蛋白水平显著上调,经MALDI-TOF-MS鉴定都是类似siderophore受体的蛋白。通过细菌荧光素酶报告基因luxAB研究相应基因的转录调控,发现它们在转录水平受缺铁诱导,并且响应缺铁诱导的表达模式存在差异。利用luxAB检测其中一些启动子的表达有可能用于水体中可利用铁离子的生物监测。

Outer membrane proteins induced by iron deficiency in Anabaena sp PCC 7120

Iron deficiency can induce cyanobacteria to synthesize siderophore receptor proteins on the outer membrane to enhance the uptake of iron. In this study, an outer membrane of high purity was prepared from Anabaena sp. PCC 7120 based on aqueous polymer two-phase partitioning and discontinuous sucrose density ultra-centrifugation, and the induction of outer membrane proteins by iron deficiency was investigated using 2-D gel electrophoresis. At least. five outer membrane proteins were newly synthesized or significantly up-regulated in cells transferred to iron-deficient conditions, which were all identified to be siderophore receptor proteins according to MALDI-TOF-MS analyses. Bacterial luciferase reporter genes luxAB were employed to monitor the transcription of the encoding genes. The genes were induced by iron deficiency at the transcriptional level in different responsive modes. Luciferase activity expressed from an iron-regulated promoter may be used as a bioreporter for utilizable iron in natural water samples. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Effects of iron on growth, pigment content, photosystem II efficiency, and siderophores production of Microcystis aeruginosa and Microcystis wesenbergii

Changes in growth, photosynthetic pigments, and photosystem II (PS II) photochemical efficiency as well as production of siderophores of Microcystis aeruginosa and Microcystis wesenbergii were determined in this experiment. Results showed growths of M. aeruginosa and M. wesenbergii, measured by means of optical density at 665 nm, were severely inhibited under an iron-limited condition, whereas they thrived under an iron-replete condition. The contents of chlorophyll-a, carotenoid, phycocyanin, and allophycocyanin under an iron-limited condition were lower than those under an iron-replete condition, and they all reached maximal contents on day 4 under the iron-limited condition. PS II photochemical efficiencies (maximal PS II quantum yield), saturating light levels (I-k ) and maximal electron transport rates (ETRmax) of M. aeruginosa and M. wesenbergii declined sharply under the iron-limited condition. The PS II photochemical efficiency and ETRmax of M. aeruginosa rose , whereas in the strain of M. wesenbergii, they declined gradually under the iron-replete condition. In addition, I-k of M. aeruginosa and M. wesenbergii under the iron-replete condition did not change obviously. Siderophore production of M. aeruginosa was higher than that of M. wesenbergii under the iron-limited condition. It was concluded that M. aeruginosa requires higher iron concentration for physiological and biochemical processes compared with M. wesenbergii, but its tolerance against too high a concentration of iron is weaker than M. wesenbergii.

Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.

Effects of Gallium-Desferrioxamine Compounds on Bacteria

Over 70% of nosocomial infections in the United States are resistant to one or more traditional antibiotics, necessitating research for alternative treatment options. This study aims to chelate gallium (Ga) onto a bacterial siderophore, desferrioxamine (DFO), to retard bacterial growth. By exploiting natural bacterial pathways, metal-siderophore treatments are hypothesized to circumvent traditional resistance mechanisms. Additionally, the GaDFO complex will be tested against several bacterial species to determine the specificity of DFO uptake. This research aims to prove the feasibility of siderophore piracy as an alternative to antibiotics. In showing the feasibility of siderophore piracy mechanisms, this research will enable the development of future avenues for protecting against resistant nosocomial infections.

tolQ is required for cloacin DF13 susceptibility in Escherichia coli expressing the aerobactin/cloacin DF13 receptor IutA

We investigated the role of the tolQ gene in the import of cloacin DF13 across the outer membrane of Escherichia coli strains expressing the IutA receptor. The IutA outer-membrane protein is the receptor for the siderophore ferric aerobactin and also binds cloacin DF13, a bacteriocin produced by strains of Enterobacter aerogenes. In this report we present evidence that tolQ is required for the internalization of cloacin DF13 upon binding to IutA but it is not involved in the transport of ferric aerobactin.

Polymorphism in the aerobactin-cloacin DF13 receptor genes from an enteroinvasive strain of Escherichia coli and pColV-K30 is associated only with a decrease in cloacin susceptibility

We have cloned chromosomal genes mediating the aerobactin iron transport system from the enteroinvasive strain Escherichia coli 978-77. The physical map of the region spanning the siderophore biosynthesis genes and the upstream portion of the receptor gene in strain 978-77-derived clones was identical to the corresponding regions in pColV-K30, while the downstream portion was different. Recombinant plasmids derived from strain 978-77 encoded a 76-kDa outer membrane protein, in contrast to the 74-kDa polypeptide encoded by similar clones derived from pColV-K30. No differences were found in the uptake of ferric aerobactin mediated by either the 76-kDa- or the 74-kDa-encoding plasmids. In contrast, cells containing the 76-kDa-encoding plasmids showed a 16-fold decrease in susceptibility to cloacin compared with cells harboring the 74-kDa-encoding plasmids. Two classes of chimeric aerobactin receptor genes were constructed by exchanging sequences corresponding to the downstream portion from the aerobactin receptor gene of both systems. The pColV-K30-978-77 chimeric gene encoded a 76-kDa outer membrane protein which mediated a low level of cloacin susceptibility, whereas the 978-77-pColV-K30 type encoded a protein of 74 kDa determining a level of cloacin susceptibility identical to that mediated by pColV-K30.

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group have shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-on-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening, that of metabolism and transport (32% of the mutants), and of enveloperelated genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression which in turn underlined the NF- κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS Opolysaccharide and T2SS mutants-induced responses were dependent on TLR2-TLR4- MyD88 activation suggested that LPS Opolysaccharide and PulA perturbed TLRdependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS Opolysaccharide and PulA T2SS could be new targets for designing new antimicrobials. Increasing TLR-governed defence responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Inoculation with Metal-Mobilizing Plant-GrowthPromoting Rhizobacterium Bacillus sp. SC2b and Its Role in Rhizoremediation

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

Fitness correlates with the extent of cheating in a bacterium.

There is growing awareness of the importance of cooperative behaviours in microbial communities. Empirical support for this insight comes from experiments using mutant strains, termed 'cheats', which exploit the cooperative behaviour of wild-type strains. However, little detailed work has gone into characterising the competitive dynamics of cooperative and cheating strains. We test three specific predictions about the fitness consequences of cheating to different extents by examining the production of the iron-scavenging siderophore molecule, pyoverdin, in the bacterium Pseudomonas aeruginosa. We create a collection of mutants that differ in the amount of pyoverdin that they produce (from 1% to 96% of the production of paired wild types) and demonstrate that these production levels correlate with both gene activity and the ability to bind iron. Across these mutants, we found that (1) when grown in a mixed culture with a cooperative wild-type strain, the relative fitness of a mutant is negatively correlated with the amount of pyoverdin that it produces; (2) the absolute and relative fitness of the wild-type strain in the mixed culture is positively correlated with the amount of pyoverdin that the mutant produces; and (3) when grown in a monoculture, the absolute fitness of the mutant is positively correlated with the amount of pyoverdin that it produces. Overall, we demonstrate that cooperative pyoverdin production is exploitable and illustrate how variation in a social behaviour determines fitness differently, depending on the social environment.

Caractérisation du système d’acquisition de fer codé par le locus iro chez Salmonella enterica sérovar Typhi

Le fer est un élément essentiel pour les bactéries. Puisqu’elles ne peuvent le synthétiser elles-mêmes, elles utilisent un ou plusieurs systèmes d’acquisition de fer afin de se le procurer dans l’environnement, ou chez l’hôte pour leurs propres métabolismes. Différentes stratégies coexistent chez les bactéries pathogènes dues à la faible concentration de cet élément, autant chez l’hôte que dans l’environnement. Salmonella enterica sérovar Typhi (S. Typhi) est une entérobactérie Gram négative causant une maladie systémique, soit la fièvre typhoïde, qui est spécifique à l’homme. Les mécanismes de pathogènese de ce sérovar sont peu connus jusqu’à ce jour, puisque son tropisme pour l’humain empêche l’utilisation d’un modèle animal adéquat. L’objectif de cette recherche est de caractériser le système d’acquisition de fer chez S. Typhi encodé par le locus iro. Les gènes du locus, iroBCDEN ont fait l’objet de plusieurs recherches chez différents pathogènes, notamment E. coli et Salmonella Typhimurium. Bien qu’un rôle dans la virulence ait été établi pour ce locus chez ces bactéries, très peu d’informations sont disponibles quant au rôle chez S. Typhi, qui emprunte plutôt la voie systémique d’infection. Nous avons évalué le rôle de la synthèse, de l’exportation et de l’importation du sidérophore salmochéline, codé par les gènes iroBCDEN. En inactivant le locus puis par la suite les gènes de façon indépendante par échange allélique, il a été possible d’observer leurs implications in vitro lors d’infections de cellules humaines. Le rôle dans l’adhésion et l’invasion des cellules épithéliales ainsi que le rôle dans la phagocytose et la survie dans les macrophages ont donc été déterminés. De plus, le mécanisme de sécrétion par lequel la salmochéline peut traverser la membrane externe est inconnu à ce jour. La pompe à efflux TolC est responsable de la sécrétion de plusieurs molécules, y compris l’entérobactine, un sidérophore analogue à la salmochéline. Par mutagénèse, nous avons effectué un mutant de délétion tolC afin de vérifier son implication dans l’interaction avec les cellules épithéliales et les macrophages. Afin de caractériser in vitro les mutants, nous avons effectué des courbes de croissance dans différents milieux. La sensibilité au peroxyde d’hydrogène a été vérifiée par la suite, puis dû aux résultats d’infections, la mobilité de la souche ΔtolC a été évaluée. Ces différents tests nous ont permis de mieux comprendre l’implication du locus iro, de ses composantes et de la pompe à efflux TolC lors de l’interaction avec les cellules cibles d’une infection systémique causée par Salmonella Typhi.

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections