Colonization Density Research Articles

Genome reannotation of Escherichia coli CFT073 with new insights into virulence

BackgroundAs one of human pathogens, the genome of Uropathogenic Escherichia coli strain CFT073 was sequenced and published in 2002, which was significant in pathogenetic bacterial genomics research. However, the current RefSeq annotation of this pathogen is now outdated to some degree, due to missing or misannotation of some essential genes associated with its virulence. We carried out a systematic reannotation by combining automated annotation tools with manual efforts to provide a comprehensive understanding of virulence for the CFT073 genome.ResultsThe reannotation excluded 608 coding sequences from the RefSeq annotation. Meanwhile, a total of 299 coding sequences were newly added, about one third of them are found in genomic island (GI) regions while more than one fifth of them are located in virulence related regions pathogenicity islands (PAIs). Furthermore, there are totally 341 genes were relocated with their translational initiation sites (TISs), which resulted in a high quality of gene start annotation. In addition, 94 pseudogenes annotated in RefSeq were thoroughly inspected and updated. The number of miscellaneous genes (sRNAs) has been updated from 6 in RefSeq to 46 in the reannotation. Based on the adjustment in the reannotation, subsequent analysis were conducted by both general and case studies on new virulence factors or new virulence-associated genes that are crucial during the urinary tract infections (UTIs) process, including invasion, colonization, nutrition uptaking and population density control. Furthermore, miscellaneous RNAs collected in the reannotation are believed to contribute to the virulence of strain CFT073. The reannotation including the nucleotide data, the original RefSeq annotation, and all reannotated results is freely available via http://mech.ctb.pku.edu.cn/CFT073/.ConclusionAs a result, the reannotation presents a more comprehensive picture of mechanisms of uropathogenicity of UPEC strain CFT073. The new genes change the view of its uropathogenicity in many respects, particularly by new genes in GI regions and new virulence-associated factors. The reannotation thus functions as an important source by providing new information about genomic structure and organization, and gene function. Moreover, we expect that the detailed analysis will facilitate the studies for exploration of novel virulence mechanisms and help guide experimental design.

The role of complement in innate and adaptive immunity to pneumococcal colonization and sepsis in a murine model

Streptococcus pneumoniae is an important bacterial cause of sepsis, meningitis, pneumonia and otitis media. Pneumococcal disease is generally preceded by mucosal colonization with the homologous strain; hence, resistance to colonization may be an important aspect of resistance to disease. In humans, complement deficiency is a risk factor for the development of pneumococcal disease. Although many studies have shown protective effects of complement during pneumonia and meningitis, there have been no studies reported that evaluate the role of complement in containment of pneumococcal colonization. To this end, we studied the role of complement in preventing the progression of pneumococcal mucosal colonization to sepsis in a mouse model. Sepsis developed in 60% of complement-depleted mice following intranasal pneumococcal challenge, but not in control or neutrophil-depleted mice. Colonization density in the nasopharynx and local mucosal tissue was similar between complement-depleted and control mice before onset of sepsis. Immunization of complement-depleted mice with an intranasally administered whole cell pneumococcal vaccine (WCV) reduced progression towards sepsis and protected surviving mice against colonization comparably to complement-sufficient mice. We therefore conclude that complement prevents sepsis following pneumococcal colonization in a neutrophil-independent fashion, but and WCV-induced adaptive immunity is complement-independent.

Candida Colonization and Candiduria in Critically Ill Patients in the Intensive Care Unit

Clinical severity of invasive candidiasis in critically ill patients and existing difficulties in timely diagnosis mean that early empirical therapy, based upon a strict clinical and epidemiological judgement, is required in intensive care unit patients. One school of thought is that the clinical severity and epidemiological burden of this disease warrant prophylaxis in all critically ill patients. In reality, however, there are still many open-ended questions with regard to which variables are most apt for selection of patients requiring prophylactic or empirical treatment. As a consequence of a consistently significant correlation between colonization (one or more Candida-positive cultures from non-sterile sites) and subsequent infection, colonization remains the most universally accepted predictive variable with regard to invasive candidiasis. This is particularly true for high density colonization. It has not yet been clarified whether colonization can be used in isolation to identify high-risk patients or if it should be combined with other variables indicating high risk. Additionally, there is still a debate surrounding the question as to whether determination of multisite colonization is required, or whether detecting colonization at one or two specific sites is sufficient for the identification of high-risk patients. From a practical perspective, candiduria (a frequent finding in critically ill patients) appears one of the most promising parameters with regard to single-site assessment, owing to easy sampling procedures. Definitive evidence of a correlation between candiduria and invasive candidiasis is currently still lacking, as the few published studies thus far have yielded conflicting results. It is, however, apparent that candiduria can be reliably considered a surrogate marker of high density of colonization, thereby potentially representing a more practical, less resource-intensive screening marker of heavy colonization and high risk of infection than is currently possible using parameters such as the multiple-site colonization index.

Regulation of Gastric B Cell Recruitment Is Dependent on IL-17 Receptor A Signaling in a Model of Chronic Bacterial Infection

Th17-driven immune responses contribute to the pathogenesis of many chronic inflammatory diseases. In this study, we investigated the role of IL-17 signaling in chronic gastric inflammation induced by Helicobacter pylori, a Gram-negative bacterium that persistently colonizes the human stomach. Wild-type C57BL/6 mice and mice lacking IL-17RA (IL-17RA(-/-)) were orogastrically infected with H. pylori. Differences in bacterial colonization density and gastric inflammation were not apparent at 1 mo postinfection, but by 3 mo postinfection, H. pylori colonization density was higher and mononuclear gastric inflammation more severe in infected IL-17RA(-/-) mice than in infected wild-type mice. A striking feature was a marked increase in gastric B cells, plasma cells, and lymphoid follicles, along with enhanced H. pylori-specific serum Ab responses, in infected IL-17RA(-/-) mice. Fewer gastric neutrophils and lower levels of neutrophil-recruiting chemokines were detected in infected IL-17RA(-/-) mice than in infected wild-type mice. Gastric IL-17a and IL-21 transcript levels were significantly higher in infected IL-17RA(-/-) mice than in infected wild-type mice or uninfected mice, which suggested that a negative feedback loop was impaired in the IL-17RA(-/-) mice. These results underscore an important role of IL-17RA signaling in regulating B cell recruitment. In contrast to many chronic inflammatory diseases in which IL-17RA signaling promotes an inflammatory response, IL-17RA signaling down-regulates the chronic mononuclear inflammation elicited by H. pylori infection.

Similarity of Mast Cell Density and Distribution in Terminal Ileum and Colon of IBD and IBS Patients and Normal Controls

Purpose: Background: In vitro and animal studies showed that Mast Cells (MC) are critical element of inflammatory cascade. However, the role of MC in IBD and IBS is still controversial. One possible reason for this controversy is lack of standard method to assess the size of gastrointestinal mucosal MC population in various region of the gastrointestinal tract. Hypoythesis: The number of MC in various part of GI tract is different among subjects with IBD, IBS in comparison to healthy controls. Aim: In this study we tried to evaluate the number of mucosal MC in subjects with IBD, IBS and compare the data with healthy controls. Methods: 24 subjects (8 Controls, 5 inactive IBD, 11 IBS) were studied. Total of 55 biopsy samples were taken during colonoscopy from sigmoid, ascending colon and ileum. Biopsies were stained using immunohistochemical techniques using monoclonal antibody against CD-117. A total of 9 random distinct fields, which were completely covered by transversely cut glands, were selected for MC counting and the mean value was considered as the number of MC per HPF. The counting was done by two independent observer. The number of MC was compared using ANOVA test. Results: There was on average 17 MC per HPF in the ileum, 16 in the ascending colon and 14 in the sigmoid colon. The number of MC in right colon was 20 in IBS-c while this number was 13 in healthy controls and 12 in subjects with IBS-d. However, this difference did not reach statistical significance (p=0.06). The number of MC in other part of the colon and ileum was not significantly different among the subjects group. The inter-observer variability was also not different among the groups. Conclusion: There was no significant difference in the number of MC in various region of lower GI tract in subjects with IBD, IBS compared to healthy counterparts. This may suggest that MC quantity may not be a determining factor in evaluating the role of MC in these disorders. Further studies that assess the function of MC may be more appropriate method of investigating the role of MC in various region of the GI tract in subjects with IBD or IBS.

Helicobacter spp. in Cats: Association between Infecting Species and Epithelial Proliferation within the Gastric Lamina Propria

The aim of this study was to determine whether there is an association between Helicobacter spp. infection of the feline stomach and the presence of gastric lesions and epithelial proliferation within the mucosa of this tissue. The study included 23 pet cats of both sexes and of varied age and breed. Eighteen of these animals were clinically normal and five had a history of chronic vomiting. Samples of the mucosa of the pyloric antrum, corpus and fundus were collected by gastroscopy. The presence of Helicobacter spp. was confirmed by polymerase chain reaction (PCR) or Warthin-Starry (WS) staining and the species of Helicobacter was determined by PCR. Mucosal lesions were evaluated by examination of sections stained by haematoxylin and eosin (HE) and epithelial proliferation was determined by enumerating nucleolar organizer regions (AgNOR). In 20 (87%) cats the presence of Helicobacter spp. was confirmed by both PCR and WS. There was no significant difference in colonization density between the different gastric regions. H. heilmannii was the most frequently identified species (17 of 20 cats), and H. felis was only identified in co-infection (two of 17 cats). One sample that was PCR positive to the genus level for Helicobacter spp. was negative for the four individual species reactions. Histological changes in the lamina propria included mild mononuclear inflammatory infiltration, the presence of lymphoid follicles, fibrosis and glandular degeneration. These changes were most severe in the pyloric antrum. There was significant association between infection with gastric Helicobacter spp. and the presence of lymphoid follicles (P=0.03), and between infection and epithelial proliferation in the antrum (P<0.01), corpus (P<0.001) and fundus (P<0.001).

Role of IL-17 receptor alpha (IL-17Rα) signaling in Helicobacter pylori-host interactions (129.7)

Abstract H. pylori persistently colonizes the human stomach and induces a gastric mucosal inflammatory response. To investigate a potential role of Th17 responses in modulating H. pylori-host interactions, wild-type C57Bl/6 (WT) mice and IL-17Rα-/- mice were orogastrically infected with H. pylori. By 3 months post-infection, H. pylori colonization density was significantly higher in IL-17Rα-/- mice than in WT mice. Gastric inflammation, assessed by histologic staining, was significantly more severe in H. pylori-infected IL-17Rα-/- mice than in infected WT mice. FACS analysis indicated that infected IL-17Rα-/- mice had significantly higher numbers of gastric CD4+ cells and B cells and significantly lower numbers of gastric neutrophils, compared to infected WT mice. IFNγ, IL-4, and multiple chemokines (KC, LIX, MIP2) were expressed at significantly higher levels in stomachs of infected WT mice than in infected IL-17Rα-/- mice. In contrast, gastric IL-17 transcript levels were 9-fold higher in infected IL-17Rα-/- mice than in infected WT mice. Primary gastric epithelial cells (PGEC) harvested from WT mice expressed KC and GM-CSF in response to recombinant IL-17, whereas cells from IL-17Rα-/- mice did not. PGECs from both WT and IL-17Rα-/- mice expressed IL-6, IL-1β, and MIP2 in response to IL-17. These data indicate that IL-17Rα signaling has an important role in modulating the gastric mucosal response to H. pylori.

General microflora, arbuscular mycorrhizal colonization and occurrence of endophytes in the rhizosphere of two age groups of Ginkgo biloba L.of Indian Central Himalaya

The populations of the general microflora (bacteria, actinomycetes and fungi) in the rhizosphere and their corresponding non-rhizosphere soil samples of Ginkgo biloba L. of two age groups (Group A, <25 years-young trees; Group B, >60 years-old trees) growing under a temperate location of Indian Himalayan Region (IHR) have been determined. Observations were also made for the diversity, distribution and colonization of arbuscular mycorrhizal (AM) fungi and occurrence of endophytes in roots of G. biloba. The population of general microflora was found to be higher in the rhizosphere of Group B trees, more clearly reflected in terms of rhizosphere: soil (R:S) ratios. Contrary to this, per cent colonization and spore densities of AM fungi were higher in the rhizosphere of Group A trees as compared to the rhizosphere of Group B. AM fungal colonization was observed mostly in form of loose coils. All the spores detected, belonged to the genus Glomus with five different types. Presence of endophytes (both bacteria and fungi) was observed in the cortical cells of G. biloba roots, more profound in case of Group B trees. Data suggest that, while the species of Glomus dominated the rhizosphere of G. biloba, an inverse correlation exist between the colonization of general microflora and the colonization of AM fungi including endophytes.

Mycorrhizal and dark septate endophytic fungi under the canopies of desert plants in Mu Us Sandy Land of China

Biodiversity of arbuscular mycorrhizal colonization and spore density was investigated in 20 desert plants (dominant or common species) collected from different locations of Mu Us Sandy Land of China. We observed three mycorrhizal types including Arum-type, Paris-type, and an intermediate type among the plants. Another type of potentially beneficial fungi associated with roots of all species was also observed, namely, dark septate endophytic fungi (DSEF). Of the 20 sample plants examined, all species were coinfected by the two target fungi (Arbuscular mycorrhizal fungi (AMF) colonization of Salix psammophila and DSEF colonization of Periploca sepium were as low as 4%). Based on this investigation, we speculated that the DSEF are ubiquitous in desert ecosystems and can co-occur with AMF in desert plants, functioning much like mycorrhizal fungi. Further studies will be required to elucidate interactional mechanisms with AMF and the mechanisms operating in desert ecosystem.

Helicobacter pylori cag-Pathogenicity Island-Dependent Early Immunological Response Triggers Later Precancerous Gastric Changes in Mongolian Gerbils

Infection with Helicobacter pylori, carrying a functional cag type IV secretion system (cag-T4SS) to inject the Cytotoxin associated antigen (CagA) into gastric cells, is associated with an increased risk for severe gastric diseases in humans. Here we studied the pathomechanism of H. pylori and the role of the cag-pathogenicity island (cag-PAI) for the induction of gastric ulcer and precancerous conditions over time (2–64 weeks) using the Mongolian gerbil model. Animals were challenged with H. pylori B128 (WT), or an isogenic B128ΔcagY mutant-strain that produces CagA, but is unable to translocate it into gastric cells. H. pylori colonization density was quantified in antrum and corpus mucosa separately. Paraffin sections were graded for inflammation and histological changes verified by immunohistochemistry. Physiological and inflammatory markers were quantitated by RIA and RT-PCR, respectively. An early cag-T4SS-dependent inflammation of the corpus mucosa (4–8 weeks) occurred only in WT-infected animals, resulting in a severe active and chronic gastritis with a significant increase of proinflammatory cytokines, mucous gland metaplasia, and atrophy of the parietal cells. At late time points only WT-infected animals developed hypochlorhydria and hypergastrinemia in parallel to gastric ulcers, gastritis cystica profunda, and focal dysplasia. The early cag-PAI-dependent immunological response triggers later physiological and histopathological alterations towards gastric malignancies.

Patterns and density of early tracheal colonization in intensive care unit patients

Objective The study aimed to describe the patterns and density of early tracheal colonization among intubated patients and to correlate colonization status with levels of antimicrobial peptides and inflammatory cytokines. Design The was a prospective cohort study. Setting The study was conducted in medical and cardiovascular intensive care units of a tertiary referral hospital. Patients Seventy-four adult patients admitted between March 2003 and May 2006 were recruited for the study. Interventions Tracheal aspirates were collected daily for the first 4 days of intubation using standardized, sterile technique and sent for quantitative culture and cytokines, lactoferrin and lysozyme measurements. Measurements and Main Results The mean acute physiology and chronic health evaluation (APACHE II) score in this cohort was 24 ± 7. Proportion of subjects colonized by any microorganism increased over the first 4 days of intubation (47%, 60%, 70%, 70%, P = .08), but density of colonization for bacteria or yeast did not change significantly. No known risk factors predicted tracheal colonization on day 1 of intubation. Several patterns of colonization were observed (persistent, transient, new colonization, and clearance of initial colonization).The most common organisms cultured were Candida albicans and coagulase-negative Staphylococcus. Levels of cytokines, lactoferrin, or lysozyme did not change over time and were not correlated with tracheal colonization status. Four subjects (6%) had ventilator-associated pneumonia. Conclusions The density of tracheal colonization did not change significantly over the first 4 days of intubation in medical intensive care unit patients. There was no correlation between tracheal colonization and the levels of antimicrobial peptides or cytokines. Several different patterns of colonization may have to be considered while planning interventions to reduce airway colonization.

Topographic position modulates the mycorrhizal response of oak trees to interannual rainfall variability

California coast live oak (Quercus agrifolia) forms tripartite symbiotic associations with arbuscular (AMF) and ectomycorrhizal (EMF) fungi. We selected oak individuals differing in topographic position and depth to groundwater (mesic valley vs. xeric hill sites) to investigate changes of tree mycorrhizal status in response to interannual rainfall variability. EMF root colonization, as well as hyphal abundance and viability in upper rhizosphere soil (0-30 cm), were negatively affected by severe multi-year drought, although not to the same extent in each topographic location. Oak trees growing in hill sites showed EMF colonization levels <1% in upper roots during drought. By contrast, oaks in valley sites maintained much higher EMF colonization (>19%) in upper roots during drought. EMF root colonization increased sharply at both topographic positions during the ensuing wet year (78% in valley, 49% in hill), which indicates that the mycorrhizal status of roots in upper rhizosphere soil is highly responsive to interannual rainfall variability. Across sites and years, percentage EMF colonization and soil hyphal density and viability were strongly positively correlated with soil moisture potential, but percentage AMF root colonization was not. Interestingly, changes in percentage EMF root colonization and density of viable hyphae between a wet and a dry year were proportionally much greater in xeric hill sites than in mesic valley sites. The mycorrhizal status of oak trees was particularly responsive to changes in soil moisture at the hill sites, where roots in upper rhizosphere soil shifted from almost exclusively AMF during severe drought to predominantly EMF during the ensuing wet year. By contrast, the mycorrhizal status of oaks in the valley sites was less strongly coupled to current meteorological conditions, as roots in upper soil layers remained predominantly EMF during both a dry and a wet year. Canopy shading and hydraulic lift by oaks in valley sites likely contributed to maintain the integrity and viability of EMF roots and extraradical hyphae in upper rhizosphere soil during extended drought. Our results suggest that oak woodlands in water-limited ecosystems may become increasingly reliant on the AMF symbiosis under future climate change scenarios for the U.S. southwest and other world regions.

Impaired Innate and Adaptive Immunity to Streptococcus pneumoniae and Its Effect on Colonization in an Infant Mouse Model

Streptococcus pneumoniae colonization and invasive disease peak around the third and first birthdays, respectively, and decline thereafter. While these declines are attributable in part to immunity acquired via natural exposure, maturation of innate immune responses may also be involved. A mucosally administered candidate whole-cell pneumococcal vaccine (WCV) containing killed pneumococcal antigen (WCA) plus a cholera toxin adjuvant protects against intranasal carriage of pneumococci by a mechanism that is antibody independent and CD4(+) TH17 cell dependent. Because infants and children are a key target population for this vaccine, we sought to evaluate the immune responses of neonatal and infant mice to S. pneumoniae and to assess whether the WCV would be effective in these mice. Like human infants, infant mice showed impaired clearance of nasopharyngeal colonization with S. pneumoniae. Macrophages from neonatal and infant mice stimulated with killed pneumococci in vitro showed significantly reduced cytokine production, including that of KC, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage chemoattractant protein 1, interleukin-6 (IL-6), IL-1alpha, tumor necrosis factor alpha, and gamma interferon, whereas IL-10 expression was significantly increased compared to that in macrophages from adult mice. IL-17A production from adult immune CD4(+) T cells was significantly delayed when neonatal macrophages instead of adult macrophages were used as antigen-presenting cells. Moreover, whole blood from mice immunized as neonates with WCV produced significantly less IL-17A after stimulation with WCA than did blood from mice immunized as adults. Nonetheless, a single immunization of neonatal mice with WCV significantly reduced colonization density. Overall, our data suggest an impairment of both innate and acquired cellular immune responses in neonatal and infant mice. However, WCV confers a significant reduction in colonization following pneumococcal challenge, suggesting that it may still be effective in the setting of immature immune responses.

GastroPanel: Evaluation of the usefulness in the diagnosis of gastro-duodenal mucosal alterations in children

Background The combined evaluation of serum pepsinogens A (PGA) and C (PGC), gastrin-17 (G17) and anti- H. pylori antibodies (anti- H. pylori)(GastroPanel) has recently been proposed as a useful aid for investigating H. pylori-associated gastric mucosal inflammation. Our aim was to evaluate whether GastroPanel can correctly classify children who need or not endoscopy (EGD). Methods GastroPanel was performed in 554 consecutive children subjected to EGD. Results PGC and anti- H. pylori were sensitive (82.5% and 73.1%) and specific (58.1% and 84.0%) indices of H. pylori infection. Antral H. pylori colonization density, inflammation and activity grades were correlated with PGC. PGC and G17 were significantly higher in children with celiac disease (14.9 ± 0.88 µg/L and 5.6 ± 0.79 pmol/L) than in controls (8.5 ± 0.38 µg/L and 2.4 ± 0.24 pmol/L). The best cut-offs to distinguish H. pylori infected children from controls were 7.45 µg/L for PGC, 4.2 pmol/L for G17, 18 U for anti- H. pylori and 25 µg/L for PGA. With these cut-offs, GastroPanel had a NPV of 89.6% and a PPV of 66.8%. Conclusions A negative GastroPanel result in children with upper abdominal non alarm symptoms, should allow the paediatrician to reasonably rule out the presence of major gastro-duodenal diseases and therefore avoid EGD.

Arbuscular and ectomycorrhizal colonization of two Eucalyptus species in semiarid Brazil

Our goal was to evaluate the mycorrhizal colonization, as well as the density of arbuscular mycorrhizal (AM) fungal spores, in Eucalyptus camaldulensis and E. grandis monocultures at 2 years in a semiarid part of Brazil. Soil and root samples were collected in 2 consecutive years. Eucalyptus camaldulensis showed varied AM colonization level according to season of sampling, and Glomus was dominant in spore numbers. Eucalyptus grandis showed dominant ectomycorrhizal (ECM) colonization and lower AM fungal spore density. Overall results suggest that E. camaldulensis has both AM and ECM dependencies, whereas E. grandis is solely ECM dependent in the monocultures.

Early-Season Aerial Adult Colonization and Ground Activity of Pea Leaf Weevil (Coleoptera: Curculionidae) in Pea as Influenced by Tillage System

The pea leaf weevil, Sitona lineatus (L.) (Coleoptera: Curculionidae), is an important pest of pea, Pisum sativum L., in northern Idaho. Previous research revealed greater relative pea leaf weevil abundance and feeding damage in peas grown using conventional-tillage compared with no-tillage practices. However, the effects of tillage practices on early season colonization and activity by the pea leaf weevil on pea are not fully understood. Aerial traps and pitfall traps were used to assess adult colonization and relative density of adult pea leaf weevil into conventional-tillage and no-tillage pea in northern Idaho during 2005 and 2006. Feeding damage to the crop also was evaluated. During both years, aerial traps captured more pea leaf weevil in May, when crop establishment and early growth occurred, than in later months. Significantly more adult pea leaf weevils were captured in aerial traps in conventional-tillage than in no-tillage plots in May of both years. Significantly more pea leaf weevil were captured in pitfall traps in conventional-tillage plots than in no-tillage plots during the period immediately after peak aerial adult colonization in late May and early June. Crop feeding damage was significantly greater in conventional-tillage than in no-tillage plots in late May and early June. The patterns suggest that more adult pea leaf weevil colonize conventional-tillage pea than no-tillage pea. Pea plants in conventional-tillage emerged earlier and were larger than those in no-tillage during the pea leaf weevil colonization period, possibly accounting for the differences in colonization rates. This leads to greater early season pea leaf weevil infestation of conventional-tillage plots at a critical period for pea development that might ultimately influence crop yield.

Early-season aerial adult colonization and ground activity of pea leaf weevil (Coleoptera: Curculionidae) in pea as influenced by tillage system.

The pea leaf weevil, Sitona lineatus (L.) (Coleoptera: Curculionidae), is an important pest of pea, Pisum sativum L., in northern Idaho. Previous research revealed greater relative pea leaf weevil abundance and feeding damage in peas grown using conventional-tillage compared with no-tillage practices. However, the effects of tillage practices on early season colonization and activity by the pea leaf weevil on pea are not fully understood. Aerial traps and pitfall traps were used to assess adult colonization and relative density of adult pea leaf weevil into conventional-tillage and no-tillage pea in northern Idaho during 2005 and 2006. Feeding damage to the crop also was evaluated. During both years, aerial traps captured more pea leaf weevil in May, when crop establishment and early growth occurred, than in later months. Significantly more adult pea leaf weevils were captured in aerial traps in conventional-tillage than in no-tillage plots in May of both years. Significantly more pea leaf weevil were captured in pitfall traps in conventional-tillage plots than in no-tillage plots during the period immediately after peak aerial adult colonization in late May and early June. Crop feeding damage was significantly greater in conventional-tillage than in no-tillage plots in late May and early June. The patterns suggest that more adult pea leaf weevil colonize conventional-tillage pea than no-tillage pea. Pea plants in conventional-tillage emerged earlier and were larger than those in no-tillage during the pea leaf weevil colonization period, possibly accounting for the differences in colonization rates. This leads to greater early season pea leaf weevil infestation of conventional-tillage plots at a critical period for pea development that might ultimately influence crop yield.

Changes in arbuscular mycorrhizal associations and fine root traits in sites under different plant successional phases in southern Brazil

Fine root morphological traits and distribution, arbuscular mycorrhizal (AM) fungi, soil fertility, and nutrient concentration in fine root tissue were compared in sites under different successional phases: grass plants, secondary forest, and mature forest in Londrina county, Paraná state, southern Brazil. Soil cores were collected randomly at the 0-10- and 10-20-cm depths in three quadrants (50 m2) in each site. Plants from the different successional stages displayed high differences in fine root distribution, fine root traits, and mycorrhizal root colonization. There were increases in the concentration of nutrients both in soil and fine roots and decrease of bulk soil density along the succession. The fine root biomass and diameter increased with the succession progress. The total fine root length, specific root length, root hair length, and root hair incidence decreased with the succession advance. Similarly, the mycorrhizal root colonization and the density of AM fungi spores in the soil decreased along the succession. Mycorrhizal root colonization and spore density were positively correlated with fine root length, specific root length, root hair length, root hair incidence, and bulk density and negatively correlated with fine root diameter and concentration of some nutrients both in soil and root tissues. Nutrient concentration in root tissue and in soil was positively correlated with fine root diameter and negatively correlated with specific root length, root hair length, and root hair incidence. These results suggest different adaptation strategies of plant roots for soil exploration and mineral acquisition among the different successional stages. Early successional stages displayed plants with fine root morphology and AM fungi colonization to improve the root functional efficiencies for uptake of nutrients and faster soil resource exploration. Late successional stages displayed plants with fine root morphology and mycorrhizal symbiosis for both a lower rate of soil proliferation and soil exploration capacity to acquire nutrients.

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

Prevotella bivia as a source of lipopolysaccharide in the vagina

Objectives To compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA). Methods Vaginal washes obtained from patients with ( n = 43) and without ( n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts. Results The median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log 10 CFU/ml, respectively, P < 0.001). Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS). The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV. Conclusion P. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.