Transposase and cointegrase: specialized transposition proteins of the bacterial insertion sequence IS21 and related elements.

The bacterial insertion sequence IS21 shares with many insertion sequences a two-step, reactive junction transposition pathway, for which a model is presented in this review: a reactive junction with abutted inverted repeats is first formed and subsequently integrated into the target DNA. The reactive junction occurs in IS21-IS21 tandems and IS21 minicircles. In addition, IS21 shows a unique specialization of transposition functions. By alternative translation initiation, the transposase gene codes for two products: the transposase, capable of promoting both steps of the reactive junction pathway, and the cointegrase, which only promotes the integration of reactive junctions but with higher efficiency. This review also includes a survey of the IS21 family and speculates on the possibility that other members present a similar transpositional specialization.

Determination of bacterial quantity by sonication in vacum-assisted closure (VAC) foams used for treatment of chronic wounds

Background: Negative pressure wound treatment is increasingly used through a Vacuum-Assisted Closure (VAC) device in complex wound situations. For this purpose, sterile polyurethane (PU) and polyvinyl alcohol (PVA) foam dressings are fitted to the wound size and covered with an adhesive drape to create an airtight seal. Little information exists about the type and quantity of microorganisms within the foams. Therefore, we investigated VAC foams after removal from the wound using a validated method (sonication) to detect the bacterial bioburden in the foam consisting as microbial biofilms.Methods: We prospectively included VAC foams (PU and PVA, KCI, Rümlamg, Switzerland) without antibacterial additions (e.g. silver), which were removed from wounds in patients with chronic ulcers from January 2007 through December 2008. Excluded were patients with acute wound infection, necrotizing fasciitis, underlying osteomyelitis or implant. Removed foams from regular changes of dressing were aseptically placed in a container with 100 ml sterile Ringer's solution. Within 4 hours after removal, foams were sonicated for 5 min at 40 kHz (as described in NEJM 2007;357:654). The resulting sonication fluid was cultured at 37°C on aerobic blood agar plates for 5 days. Microbes were quantified as No. of colony-forming units (CFU)/ml sonication fluid and identified to the species level.Results: A total of 68 foams (38 PU and 30 PVA) from 55 patients were included in the study (median age 71 years; range 33-88 years, 57% were man). Foams were removed from the following anatomic sites: sacrum (n=29), ischium (n=18), heel (n=13), calves (n=6) and ankle (n=2). The median duration of being in place was 3 days (range, 1-8 days). In all 68 foams, bacteria were found in large quantities (median 105 CFU/ml, range 102-7 CFU/ml sonication fluid. No differences were found between PU and PVA foams. One type of organisms was found in 11 (16%), two in 17 (24%) and 3 or more in 40 (60%) foams. Gram-negative rods (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa) were isolated in 70%, followed by Staphylococcus aureus (20%), koagulase-negative staphylococci, streptococci (8%), and enterococci (2%).Conclusion: With sonication, a high density of bacteria present in VAC foams was demonstrated after a median of 3 days. Future studies are needed to investigate whether antimicrobial-impregnated foams can reduce the bacterial load in foams and potentially improve wound healing.

Iowa's Water - Ambient Monitoring Program: Bacterial Monitoring of Iowa's Beaches, January 2001

The Iowa Department of Natural Resources has produced an 4 page article about how to assess Iowa's streams and rivers. How to use ambient monitoring of streams and river in Iowa.

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Bacterial transcriptional regulators for degradation pathways of aromatic compounds.

Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways.

Protection from lethal gram-negative bacterial sepsis by targeting Toll-like receptor 4.

Toll-like receptor 4 (TLR4), the signal-transducing molecule of the LPS receptor complex, plays a fundamental role in the sensing of LPS from gram-negative bacteria. Activation of TLR4 signaling pathways by LPS is a critical upstream event in the pathogenesis of gram-negative sepsis, making TLR4 an attractive target for novel antisepsis therapy. To validate the concept of TLR4-targeted treatment strategies in gram-negative sepsis, we first showed that TLR4(-/-) and myeloid differentiation primary response gene 88 (MyD88)(-/-) mice were fully resistant to Escherichia coli-induced septic shock, whereas TLR2(-/-) and wild-type mice rapidly died of fulminant sepsis. Neutralizing anti-TLR4 antibodies were then generated using a soluble chimeric fusion protein composed of the N-terminal domain of mouse TLR4 (amino acids 1-334) and the Fc portion of human IgG1. Anti-TLR4 antibodies inhibited intracellular signaling, markedly reduced cytokine production, and protected mice from lethal endotoxic shock and E. coli sepsis when administered in a prophylactic and therapeutic manner up to 13 h after the onset of bacterial sepsis. These experimental data provide strong support for the concept of TLR4-targeted therapy for gram-negative sepsis.

Genome-Wide Prediction Methods in Highly Diverse and Heterozygous Species: Proof-of-Concept through Simulation in Grapevine.

Nowadays, genome-wide association studies (GWAS) and genomic selection (GS) methods which use genome-wide marker data for phenotype prediction are of much potential interest in plant breeding. However, to our knowledge, no studies have been performed yet on the predictive ability of these methods for structured traits when using training populations with high levels of genetic diversity. Such an example of a highly heterozygous, perennial species is grapevine. The present study compares the accuracy of models based on GWAS or GS alone, or in combination, for predicting simple or complex traits, linked or not with population structure. In order to explore the relevance of these methods in this context, we performed simulations using approx 90,000 SNPs on a population of 3,000 individuals structured into three groups and corresponding to published diversity grapevine data. To estimate the parameters of the prediction models, we defined four training populations of 1,000 individuals, corresponding to these three groups and a core collection. Finally, to estimate the accuracy of the models, we also simulated four breeding populations of 200 individuals. Although prediction accuracy was low when breeding populations were too distant from the training populations, high accuracy levels were obtained using the sole core-collection as training population. The highest prediction accuracy was obtained (up to 0.9) using the combined GWAS-GS model. We thus recommend using the combined prediction model and a core-collection as training population for grapevine breeding or for other important economic crops with the same characteristics.

Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation.

Although Drosophila systemic immunity is extensively studied, little is known about the fly's intestine-specific responses to bacterial infection. Global gene expression analysis of Drosophila intestinal tissue to oral infection with the Gram-negative bacterium Erwinia carotovora revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. Ingestion of bacteria had a dramatic impact on the physiology of the gut that included modulation of stress response and increased stem cell proliferation and epithelial renewal. Our data suggest that gut homeostasis is maintained through a balance between cell damage due to the collateral effects of bacteria killing and epithelial repair by stem cell division. The Drosophila gut provides a powerful model to study the integration of stress and immunity with pathways associated with stem cell control, and this study should prove to be a useful resource for such further studies.

Correlation between placental bacterial culture results and histological chorioamnionitis: a prospective study on 376 placentas.

OBJECTIVE: To study the correlation between the bacteriological and histopathological findings in placentas from women with suspected or proven chorioamnionitis (CA). METHODS: Over a 1-year period, 376 placentas were prospectively collected and processed for bacteriological and pathological studies in cases of confirmed or suspected maternal or neonatal infection. RESULTS: Histological CA was diagnosed in 26.9% of placentas (101/376), and 27.7% (28/101) of these placentas had positive bacteriological cultures. A monomicrobial culture, mainly represented by Gram-positive cocci and Gram-negative bacilli, was identified in 27% of the positive bacterial cultures. The proportion of positive cultures was higher (p=0.03) when CA was associated with funisitis, as compared with placental samples with early CA. In placentas without histological CA, bacteriological cultures were mostly negative (230/275), although pathogenic bacteria were identified in 16.3% of them (45/275). CONCLUSIONS: The histological and bacteriological results were concordant in about 70% of the examined placentas, with 61.1% negative cases (CA absent and negative bacterial cultures), and only 7.4% placentas with positive histological and bacteriological results. Discordant results (positive histology with negative bacteriology) were obtained in placentas with early CA documented by histology although possibly in relation with antibiotic prophylaxis and the presence of fastidious bacteria. Conversely, negative histology with positive bacteriology could be explained by the presence of an early-stage bacterial infection that has not yet led to detectable microscopic lesions.

Complete Nucleotide Sequence of Mouse Mammary Tumor Virus from JYG Chinese Wild Mice: Absence of Bacterial Insertion Sequences in the Cloned Viral gag Gene.

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.

Mesenteric fat-control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

Improved statistical analysis of low abundance phenomena in bimodal bacterial populations.

Accurate detection of subpopulation size determinations in bimodal populations remains problematic yet it represents a powerful way by which cellular heterogeneity under different environmental conditions can be compared. So far, most studies have relied on qualitative descriptions of population distribution patterns, on population-independent descriptors, or on arbitrary placement of thresholds distinguishing biological ON from OFF states. We found that all these methods fall short of accurately describing small population sizes in bimodal populations. Here we propose a simple, statistics-based method for the analysis of small subpopulation sizes for use in the free software environment R and test this method on real as well as simulated data. Four so-called population splitting methods were designed with different algorithms that can estimate subpopulation sizes from bimodal populations. All four methods proved more precise than previously used methods when analyzing subpopulation sizes of transfer competent cells arising in populations of the bacterium Pseudomonas knackmussii B13. The methods' resolving powers were further explored by bootstrapping and simulations. Two of the methods were not severely limited by the proportions of subpopulations they could estimate correctly, but the two others only allowed accurate subpopulation quantification when this amounted to less than 25% of the total population. In contrast, only one method was still sufficiently accurate with subpopulations smaller than 1% of the total population. This study proposes a number of rational approximations to quantifying small subpopulations and offers an easy-to-use protocol for their implementation in the open source statistical software environment R.

Bacterial and viral superantigens: roles in autoimmunity?

Superantigens are bacterial, viral, or retroviral proteins which can activate specifically a large proportion of T cells. In contrast with classical peptide antigen recognition, superantigens do not require processing to small peptides but act as complete or partially processed proteins. They can bind to major histocompatibility complex class II molecules and stimulate T cells expressing particular T cell receptor V beta chains. The other polymorphic parts of the T cell receptor, which are crucial for classical antigen recognition, are not important for this interaction. When this strategy is used a large proportion of the host immune system can be activated shortly after infection. The activated cells have a wide variety of antigen specificities. The ability to stimulate polyclonal B (IgG) as well as T cell responses raises possibilities of a role for superantigens in the induction of autoimmune diseases. Superantigens have been a great tool in the hands of immunologists in unravelling some of the basic mechanisms of tolerance and immunity.