Microbial Dissemination Research Articles

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4lps-d), mice deficient in TLR9 (TLR9−/−) and TLR4/9 double mutant mice (TLR4lps-d/TLR9−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4lps-d/TLR9−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4lps-d/TLR9−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.

Brain abscesses in a patient with a patent foramen ovale: a case report

IntroductionBrain abscesses arising from right-to-left cardiac shunting are very rare in adults.Case presentationWe describe the case of a 47-year-old non-hispanic white male with periodontal disease who developed several brain abscesses caused by Streptococcus intermedius. A comprehensive workup revealed a patent foramen ovale with oral flora as the only plausible explanation for the brain abscesses.ConclusionBased on this case and the relevant literature, we suggest an association between a silent patent foramen ovale, paradoxical microbial dissemination to the brain, and the development of brain abscesses.

Simian immunodeficiency virus–induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut

Salmonella typhimurium causes a localized enteric infection in immunocompetent individuals, whereas HIV-infected individuals develop a life-threatening bacteremia. Here we show that simian immunodeficiency virus (SIV) infection results in depletion of T helper type 17 (TH17) cells in the ileal mucosa of rhesus macaques, thereby impairing mucosal barrier functions to S. typhimurium dissemination. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was dominated by TH17 responses, including the expression of interleukin-17 (IL-17) and IL-22. TH17 cells were markedly depleted in SIV-infected rhesus macaques, resulting in blunted TH17 responses to S. typhimurium infection and increased bacterial dissemination. IL-17 receptor-deficient mice showed increased systemic dissemination of S. typhimurium from the gut, suggesting that IL-17 deficiency causes defects in mucosal barrier function. We conclude that SIV infection impairs the IL-17 axis, an arm of the mucosal immune response preventing systemic microbial dissemination from the gastrointestinal tract.

Fluorescent DNA probes: Study of mechanisms of changes in spectral properties and features of practical application

Spectral data and nucleic acid complex formation properties for more than 30 both newly synthesized and widely used fluorescent nucleotide-specific compounds of various classes have been analyzed. These include phenylbenzene, bisbenzimidazole, psoralen, angelicin, tetrahydrocarbazole, oxophenoxazine, and others. The main rules of a generalized model adequately explaining changes in fluorescent properties of synthetic, low molecular weight nucleotide-specific dyes depending on their chemical structure, mode of interaction with substrate, properties of assay medium, etc. are proposed. Fluorescent nucleotide-specific dyes have been originally used in newly developed methods for: express evaluation of "generalized microbial dissemination" of liquid media; evaluation of possible genotoxic effects of various foodstuffs, pharmaceutical drugs, hazardous environmental factors (including their combined effects on living organisms), etc.

Fibrin and fibrinolysis in infection and host defense.

Bacterial pathogens have frequently evolved and maintained the capacity to engage and/or activate hemostatic system components of their vertebrate hosts. Recent studies of mice with selected alterations in host plasminogen and other hemostatic factors have begun to reveal a seminal role of bacterial plasminogen activators and fibrin clearance in microbial pathogenesis. Bacterial pathogens appear to exploit host plasmin-mediated proteolysis to both support microbial dissemination and evade innate immune surveillance systems. The contribution of bacterial plasminogen activation to the evasion of the inflammatory response is particularly conspicuous with the plague agent, Yersinia pestis. Infection of control mice with wild-type Y. pestis leads to the formation of widespread foci containing massive numbers of free bacteria with little inflammatory cell infiltrate, whereas the loss of either the bacterial plasminogen activator, Pla, or the elimination of host plasminogen results in the accumulation of robust inflammatory cell infiltrates at sites of infection and greatly improved survival. Interestingly, fibrin(ogen) deficiency undermines the local inflammatory response observed with Pla-deficient Y. pestis and effectively eliminates the survival benefits posed by the elimination of either host plasminogen or bacterial Pla. These studies, and complementary studies with other human pathogens, illustrate that plasminogen and fibrinogen are extremely effective modifiers of the inflammatory response in vivo and critical determinants of bacterial virulence and host defense. Detailed studies of the inflammatory response in mice with genetically-imposed modifications in coagulation and fibrinolytic factors underscore the regulatory crosstalk between the hemostatic and immune systems.

Anti-inflammatory pathways as a host evasion mechanism for pathogens

Lipoxins play a key role in controlling potent pro-inflammatory responses triggered by infection with pathogens, such as Toxoplasma gondii and Mycobacterium tuberculosis. In order to contain microbial dissemination, infected hosts must mount a powerful immune response to prevent mortality. The onset of the chronic phase of infection is characterized by continuous cell-mediated immunity. Such potent responses are kept under tight control by a class of anti-inflammatory eicosanoids, the lipoxins. Here, we review such immune-containment strategies from the host's perspective, to keep pro-inflammatory responses under control during chronic disease, as well as from the perspective of the pathogen, which pirates the host's lipoxygenase machinery to its own advantage as a probable immune-escape mechanism.

Chronic maternal and fetal Porphyromonas gingivalis exposure during pregnancy in rabbits

This study was undertaken to develop a rabbit model of maternal exposure to Porphyromonas gingivalis and determine whether fetal or placental exposure occurs. Subcutaneous steel chambers were implanted in 8 New Zealand White female rabbits. On day 7 of pregnancy, 4 rabbits were inoculated through the chamber with 5 x 10 8 CFU/mL live P gingivalis , and 4 rabbits with broth (controls) and sacrificed at term. Polymerase chain reaction was used to detect P gingivalis in maternal and fetal liver and placenta. Fisher exact test was used to compare P gingivalis detection between groups. Among exposed does, P gingivalis was detected in 33% of the maternal livers, 49% of placentas, and 34% fetal livers compared with none from controls ( P < .001). Chronic maternal exposure to P gingivalis results in systemic dissemination, transplacental passage, and fetal exposure. This model may be useful to study placental and fetal effects of this oral pathogen and to study microbial dissemination across the placenta.

More than one type of transglutaminase in invertebrates? A second type of transglutaminase is involved in shrimp coagulation

Coagulation (clot formation) forms a physical barrier to prevent the loss of body fluid and dissemination of microbes into the haemocoel after injury or infection. Its quickness and efficiency are essential for the survival of invertebrates that rely solely on innate immunity. Transglutaminase (TG) catalyses intermolecular or intramolecular ε-(γ-glutamyl) lysine bond formation, resulting in a protein polymerisation, and plays a role in blood coagulation and post-translational protein remodelling. In the present study, we cloned a TG from shrimp ( Penaeus monodon) haemocyte cDNA. It was assigned as shrimp transglutaminase II (STG II). The STG II cDNA consists of a coding region of 2,274 bp. The deduced protein has 757 amino acid residues with a calculated molecular mass of 85,000 Da and an isoelectric point of 5.48. RT-PCR results showed a significant level of STG II expression in haemocytes but not in hepatopancreas, in contrast to shrimp STG I (AY074924.1). The genetic distance between STG II and STG I is much larger than the distance between STG II and the TG of the kuruma shrimp ( Marsupenaeus japonicus). Evidence based on tissue distribution and genetic distance suggests that no less than two types of shrimp TG exist that are encoded at different chromosomal locations. The recombinant STG II (rSTG II) incorporated a TG-specific substrate, dansylcadaverine (DCA), into clottable proteins (CP) in a calcium dependent manner. Other haemocyte- or plasma-derived TG substrate is not required for CP polymerisation but may be necessary for stable clot formation. The rSTG II catalysed clottable proteins into a long chain under transmission electron microscopy (TEM) observation. In conclusion, STG II is characterized as a haemocyte TG and is involved in coagulation.

Perspectives on the use of organic acids and short chain fatty acids as antimicrobials

Organic acids have a long history of being utilized as food additives and preservatives for preventing food deterioration and extending the shelf life of perishable food ingredients. Specific organic acids have also been used to control microbial contamination and dissemination of foodborne pathogens in preharvest and postharvest food production and processing. The antibacterial mechanism(s) for organic acids are not fully understood, and activity may vary depending on physiological status of the organism and the physicochemical characteristics of the external environment. An emerging potential problem is that organic acids have been observed to enhance survivability of acid sensitive pathogens exposed to low pH by induction of an acid tolerance response and that acid tolerance may be linked to increased virulence. Although this situation has implications regarding the use of organic acids, it may only apply to circumstances in which reduced acid levels have induced resistance and virulence mechanisms in exposed organisms. Evaluating effectiveness of organic acids for specific applications requires more understanding general and specific stress response capabilities of foodborne pathogens. Development and application of molecular tools to study pathogen behavior in preharvest and postharvest food production environments will enable dissection of specific bacterial genetic regulation involved in response to organic acids. This could lead to the development of more targeted strategies to control foodborne pathogens with organic acids.

Hypoxia and extraintestinal dissemination of Candida albicans yeast forms.

Candida albicans is a pleomorphic fungus with budding yeast and filamentous forms, and is a frequent cause of complicating infections in patients who are postsurgical, in shock, and have trauma. Many cases of systemic candidiasis are thought to orginate from the intestine, but it is unclear if the filament or the yeast is the more invasive form. Because C. albicans is relatively noninvasive and because mesenteric ischemia is thought to facilitate extraintestinal microbial dissemination, wild-type C. albicans CAF2 and mutant HLC54 (defective in filament formation) were orally inoculated into antibiotic-treated mice that were housed exclusively in room air, or were intermittently exposed to 10% oxygen for 1-h intervals. Both strains of C. albicans colonized the cecum in similar numbers (approximately 10(6.7)/g). C. albicans translocation to the draining mesenteric lymph nodes was not detected in mice inoculated with CAF2 (normoxic or hypoxic) or in normoxic mice inoculated with HLC54, but was detected in 33% (P < 0.01) of hypoxic mice inoculated with HLC54. Using Caco-2 and HT-29 enterocytes cultivated on plastic dishes and pretreated for 48 h in 10% oxygen, adherence of C. albicans HLC54 was decreased compared with wild-type CAF2, and hypoxia had no noticeable effect on adherence of either CAF2 or HLC54. Using enterocytes cultivated on permeable 8-microm filters, transepithelial migration of C. albicans CAF2 and HLC54 appeared similar. Thus, C. albicans HLC54 (defective in filament formation) was more invasive in hypoxic mice compared with wild-type CAF2, and host factors (e.g., mesenteric ischemia) rather than an innate ability to interact with enterocytes might play a more important role in extraintestinal dissemination of C. albicans yeast forms.

CD85/LIR-1/ILT2 and CD152 (Cytotoxic T Lymphocyte Antigen 4) Inhibitory Molecules Down-Regulate the Cytolytic Activity of Human CD4+T-Cell Clones Specific forMycobacterium tuberculosis

Antigen-specific cytolytic CD4+ T lymphocytes control Mycobacterium tuberculosis infection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+ T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+ cells and activation of cytolytic CD4+ cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+ cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+ T-cell clones specific for M. tuberculosis were produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased by blockade and decreased by cross-linking of the receptors. These results indicate that CD152 and CD85/LIR-1/ILT2 play a role in the regulation of the antigen-specific activity of CD4+ cytolytic T lymphocytes against PPD-presenting cells.

Controlling microbial contamination on beef and lamb meat during processing.

The microbiological quality of carcases, meat and environmental surfaces was evaluated in commercial boning rooms processing beef and lamb. There was considerable variation in the level of microbial contamination on both carcases and meat, with counts ranging from less than 20 to 10(8)/cm2 on carcases and to 2 x 10(7)/cm2 on meat. The level of microbial contamination on meat was influenced by the level of carcase contamination at boning and by the boning process itself. Carcase contamination was the major determinant of microbiological quality, as more than 70% of carcase had microbial counts greater than 10(3)/cm2. Cutting boards were a major source for microbial dissemination during boning, particularly when carcase counts were less than 10(3)/cm2. If carcases were heavily contaminated, the contamination of processing surfaces was irrelevant in determining microbial loads on meat. Where carcase contamination was at low to moderate levels, the contribution of the boning process to the contamination on meat assumed increased significance. Under these conditions, improved sanitation of cutting surfaces in the boning room resulted in a significant reduction in microbial contamination on the surface of meat. These results can form the basis for ensuring that improvements made in carcase management before boning, to improve microbiological quality, will be preserved through attention to cutting board hygiene during boning.

Immunity to intracellular bacteria.

Intracellular bacteria are endowed with the capacity to survive and replicate inside mononuclear phagocytes (MP) and, sometimes, within certain other host cells. MP are potent effectors cells that are able to engulf and kill many bacterial invaders. Therefore, intracellular bacteria had to exploit potent evasion mechanisms that allow their survival in this hostile environment. At the early phase, natural killer cells activate antibacterial defense mechanisms. During intracellular persistence, microbial proteins are processed and presented, thus initiating T cell activation. By secreting interleukins, CD4 alpha/beta TH1 cells activate MP, converting them from a habitat to a potent effector cell; TH2-dependent activities seem to be of minor importance. Cytolytic CD8 T cells represent a further element of protection. In the case of Listeria monocytogenes, the gene products responsible for virulence and for the introduction of antigens into the MHC class I pathway are being characterized. Increasing evidence points to a role of gamma/delta T lymphocytes in antibacterial immunity, although their precise function remains to be elucidated. Protection in the host is a local event focussed on granulomatous lesions. MP accumulate at the site of microbial growth and become activated through the CD4 T cell-interleukin-MP axis. Lysis of incapacitated MP and other host cells by CD8 T cells allows release and subsequent uptake by more efficient phagocytes, thus contributing to host protection. At the same time, lysis of host cells promotes microbial dissemination and causes tissue injury, which represent pathogenic aspects of the same mechanism. Research on the immune response against intracellular bacteria not only helps us to better understand how the immune system deals with "viable antigens" in constant trans-mutation, it also forms the basis for the rational design of control measures for major health problems.

Characteristics of the antilysozyme activity ofStaphylococcus aureus in different types of experimental infection

The connection between the level of Staphylococcus Aureus anti-lysozyme activity (ALA) and the character of the experimental infection course has been revealed. The subsiding type of infection was developed by infecting by clones with the low ALA and at the same time there was a shift in the structure of population at the final stage of infectious process to the reduction of its heterogeneity in respect of ALA and the preservation of the colonies only with the zero and low ALA. The protracted form of infection was provoked by the clone with the high ALA and the dynamics of microbial dissemination had rhythmical nature and a high heterogeneity in respect of ALA in the structure of population of staphylococcus was preserved and the selection of colonies with the high ALA was observed. ALA of staphylococcus is an important link of pathogenesis of persistence as it determines the duration and dynamics of the survival of the pathogen in the organism.

A new perspective of microbial survival and dissemination in a prospectively contaminated air-fluidized bed model

A major concern of users of air-fluidized beds has been the possibility that such beds might be a source of microbial contamination. The purpose of this series of prospective, controlled experiments was to measure quantitatively the dissemination and survival of Bacillus subtilis, a species of bacterium that forms desiccation-resistant spores, as it was associated with circulating and clumped microbeads after challenge in an air-fluidized bed operating under decontamination conditions of heating at 48°C and microbead agitation by an air flow of 100% at 110 cu ft/min. Microbead samples collected after B. subtilis challenge from predesignated depths and locations within the air-fluidized bed at 0.25, 1, 2, 4, 24, and 48 hours were assayed for colony-forming units (CFU) of challenge bacteria by end point dilution and streak-plate assays. Results of three experiments with an average challenge of 1110 CFU of B. subtilis per gram of microbeads indicated that no more than 0.46 CFU of B. subtilis per gram of circulating microbeads and 0.78 CFU per gram of clumped microbeads were detected after 24 to 48 hours at decontamination conditions. Few microbes were detected during three control (sterile water) challenges of the air-fluidized bed operating at 33°C with 95% to 100% air flow. This study has demonstrated that an air-fluidized bed operating at the described decontamination conditions for 24 to 48 hours caused significant decreases, averaging a thousandfold per gram, in levels of microbead-associated challenge bacteria. These results suggest that mechanical abrasive forces between microbeads at high air flows, together with heat and desiccation, may be important in the air-fluidized bed in killing microorganisms from environmental, iatrogenic, and patient sources.

Inter- and intraspecific feeding selectivity of Folsomia candida (Willem) (Collembola, Isotomidae) on fungi

Selective grazing of fungi by soil microarthropods may affect decomposition rates of litter materials and the structure of microarthropod and fungal communities. We developed laboratory methods to assay feeding selectivity and investigated the preferences of the collembolan Folsomia candida on three fungi: Acremonium sp., Paecilomyces varioti, and Penicillium citrinum. F. candida showed stronger preference for Acremonium sp. than for P. varioti and P. citrinum. Oviposition site selection followed the same pattern. Actively metabolizing hyphae of Acremonium sp. and P. varioti were preferred over senescent hyphae, while spores of P. citrinum were preferred over active hyphae. If microarthropod preference for active hyphae is extensive, microarthropod regulation of decomposition could be more important than their biomass indicates. Furthermore, as the P. citrinum results indicate, mechanisms of microbial dissemination may include selective grazing.

Endogenous microbial dissemination following severe burns in rats

This study suggests that damage to the intestinal lining, an important mechanical harrier against bacterial invasion, is the main factor leading to the dissemination of endogenous microbes. Reduced phagocytic activity of Kupffer cells and compromised opsonic function also appear to contribute to the process.

Microbial survival and dissemination associated with an air-fluidized therapy unit

Clinitron Therapy Units (CTU) are used as adjuncts to the treatment of a variety of medical conditions, including burns, decubitus ulcers, and pain.l Some patients who could benefit from air-fluidized therapy, however, may also be incontinent or have infected wounds. For this reason there is concern that the CTU could become a vehicle for the transmission of infectious agents, particularly via the aerosolization route. The Clinitron bed (SSI Corp., Charleston, SC.) is an air-fluidized therapy unit consisting of a tank filled with 681.8 kg of minute (50 to 1.50 pm diameter) silicone-coated glass beads. A compressor pump filters air through a diffuser board and up through the beads, causing the beads to become fluidized. Clumps of beads form in the presence of fluids (e.g., patient exudates) and sink to the bottom of the tank because of their increase in weight. A mesh net that lines the sides and bottom of the tank is used to sieve out the clumps. A polyfilament polyester filter sheet covers the unit, allowing the upward movement of air while containing the beads in the tank. It has been proposed that desiccation of microbial cells results from the constant upward

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: Preliminary observations with heat-derived extracts of Limulus amoebocyte lysate

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.

SANITATION IN THE SPACE AGE

The classic concepts of environmental sanitation have become an integral part of our way of life in this age. The advent of the space age requires an expansion of these concepts. We are faced today with phenomenal advances in transportation and communication. Are our sanitary surveillance techniques and regulations, basically geared for parochial communities, adequate to maintain the public health in a world served by supersonic carriers? Is our knowledge and training sufficient to meet the demands for ultraclean environments imposed by advances in medicine and industrial technology? We must be ready to consider such new problems as cleaning and disinfecting the hardware and materials being developed for space travel and exploration. We must gain a perspective of the environmental health problems of people confined in sealed capsules for long time periods. We must start thinking about the control of exotic and as yet undiscovered disease agents from other planets, the control of microbial dissemination from our world to others, the logistics of interplanetary quarantine. In addition to all of this, we must continue to provide the inhabitants of this world with a safe, clean and healthy environment.