Antibiotic therapy, endotoxin concentration in cerebrospinal fluid, and brain edema in experimental Escherichia coli meningitis in rabbits

We investigated the effect of cefotaxime and chloramphenicol on endotoxin concentrations in cerebrospinal fluid (CSF) and on the development of brain edema in rabbits with Escherichia coli meningitis. Both antibiotics were similarly effective in reducing bacterial titers. Cefotaxime, but not chloramphenicol, induced a marked increase of endotoxin in CSF, from log10 1.5 +/- 0.8 to log10 2.8 +/- 0.7 ng/ml (P less than .01). This result was associated with an increase in brain water content (405 +/- 12 g of water/100 g of dry weight compared with 389 +/- 8 g in untreated controls; P less than .01), whereas in animals treated with chloramphenicol, brain water content was identical to controls. The cefotaxime-induced increase in endotoxin concentration and brain edema were both neutralized by polymyxin B, which binds to the lipid A moiety of endotoxin, or by a monoclonal antibody to lipid A. These results indicate that treating gram-negative bacillary meningitis with selected antibiotics induces increased endotoxin concentrations in CSF that are associated with brain edema.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

Potent and broad-spectrum antimicrobial activity of CXCL14 suggests an immediate role in skin infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.

Autoantibodies against bactericidal/permeability-increasing protein (BPI) in children with acute pneumonia

Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein (BPI), an inhibitor of a lipopolysaccharide of gram-negative bacteria, are a common feature of chronic neutrophilic inflammatory processes such as cystic fibrosis. We investigated whether serum and salivary anti-BPI autoantibodies also appear in the course of acute pneumonia in 24 otherwise healthy children. Nine (38%) and four (17%) patients had detectable serum anti-BPI immunoglobulin G (IgG) (> or =4 IU mL(-1)) and IgA (ratio> or =1.2), respectively, on the day of hospital admission (day 0). There was no increase in the rate of occurrence or the concentration of these antibodies in the convalescent sera obtained on day 30. The presence of anti-BPI IgG on admission did not correlate with inflammatory markers (peripheral white blood cell count, C-reactive protein) or temperature on admission. Also, salivary anti-BPI IgA, determined on days 0, 3-5 and 30, did not appear during the course of acute pneumonia. In summary, a substantial proportion of previously healthy children have pre-existing anti-BPI IgG autoantibodies. Acute neutrophilic infection, i.e. pneumonia, however, neither triggered the appearance of new antibodies nor boosted the concentrations of pre-existing ones. Thus, in typical acute pneumonia in children, autoantibodies directed against BPI may not have clinical significance.

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic Delta asaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The Delta asaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.

Pseudomonas chlororaphis subsp. piscium subsp. nov., isolated from freshwater fish

Gram-negative, aerobic, motile, rod-shaped bacteria were isolated from the intestines of freshwater fish on two separate occasions. Colonies of both strains, JF3835(T) and JF4413, produced non-diffusible green pigment following 4-5 days incubation on Luria-Bertani agar. The most abundant fatty acids were summed feature 3 (comprising C(16 : 1)ω7c and/or C(15 : 0) iso 2-OH), C(16 : 0) and C(18 : 1)ω7c. The DNA G+C content was 62.9 mol%. Sequence analysis of the 16S rRNA gene indicated 100 % sequence similarity between the two strains. In comparison with recognized species, the new strains exhibited the greatest degree of sequence similarity with members of the Pseudomonas chlororaphis subspecies: P. chlororaphis subsp. chlororaphis (99.84 %), P. chlororaphis subsp. aurantiaca (99.75 %) and P. chlororaphis subsp. aureofaciens (99.40 %). While DNA-DNA relatedness confirmed the placement of strains JF3835(T) and JF4413 as members of the species P. chlororaphis, multilocus sequencing indicated that the strains formed a distinct cluster within it. On the basis of genotypic and phenotypic evidence, strains JF3835(T) and JF4413 represent a novel subspecies of the species P. chlororaphis, for which the name Pseudomonas chlororaphis subsp. piscium subsp. nov. is proposed. The type strain is JF3835(T) (=NCIMB 14478(T)=DSM 21509(T)).

Nicoletella semolina gen. nov., sp. nov., a new member of Pasteurellaceae isolated from horses with airway disease

Gram-negative, nonmotile bacteria that are catalase, oxidase, and urease positive are regularly isolated from the airways of horses with clinical signs of respiratory disease. On the basis of the findings by a polyphasic approach, we propose that these strains be classified as Nicoletella semolina gen. nov, sp. nov., a new member of the family Pasteurellaceae. N. semolina reduces nitrate to nitrite but is otherwise biochemically inert; this includes the lack of an ability to ferment glucose and other sugars. Growth is fastidious, and the isolates have a distinctive colony morphology, with the colonies being dry and waxy and looking like a semolina particle that can be moved around on an agar plate without losing their shape. DNA-DNA hybridization data and multilocus phylogenetic analysis, including 16S rRNA gene (rDNA), rpoB, and infB sequencing, clearly placed N. semolina as a new genus in the family Pasteurellaceae. In all the phylogenetic trees constructed, N. semolina is on a distinct branch displaying approximately 5% 16S rDNA, approximately 16% rpoB, and approximately 20% infB sequence divergence from its nearest relative within the family Pasteurellaceae. High degrees of conservation of the 16S rDNA (99.8%), rpoB (99.6%), and infB (99.7%) sequences exist within the species, indicating that N. semolina isolates not only are phenotypically homogeneous but also are genetically homogeneous. The type strain of N. semolina is CCUG43639(T) (DSM16380(T)).

Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.

RTX toxins in Pasteurellaceae

RTX toxins (repeats in the structural toxin) are pore-forming protein toxins produced by a broad range of pathogenic Gram-negative bacteria. In vitro, RTX toxins mostly exhibit a cytotoxic and often also a hemolytic activity. They are particularly widespread in species of the family Pasteurellaceae which cause infectious diseases, most frequently in animals but also in humans. Most RTX toxins are proteins with a molecular mass of 100-200 kDa and are post-translationally activated by acylation via a specific activator protein. The repeated structure of RTX toxins, which gave them their name, is composed of iterative glycine-rich nonapeptides binding Ca2+ on the C-terminal half of the protein. Genetic analysis of RTX toxins of various species of Pasteurellaceae and of a few other Gram-negative bacteria gave evidence of horizontal transfer of genes encoding RTX toxins and led to speculations that RTX toxins might have originated from Pasteurellaceae. The toxic activities of RTX toxins in host cells may lead to necrosis and apoptosis and the underlying detailed mechanisms are currently under investigation. The impact of RTX toxins in pathogenicity and the immune responses of the host were described for several species of Pasteurellaceae. Neutralizing antibodies were shown to significantly reduce the cytotoxic activity of RTX toxins. They constitute a valuable strategy in the development of immuno-prophylactics against several animal diseases caused by pathogenic species of Pasteurellaceae. Although many RTX toxins possess cytotoxic and hemolytic activities toward a broad range of cells and erythrocytes, respectively, a few RTX toxins were shown to have cytotoxic activity only against cells of specific hosts and/or show cell-type specificity. Further evidence exists that RTX toxins play a potential role in host specificity of certain pathogens.

The Fascinating Coat Surrounding Mycobacteria

The mycobacterial cell envelope is fascinating in several ways. First, its composition is unique by the exceptional lipid content, which consists of very long-chain (up to C90) fatty acids, the so-called mycolic acids, and a variety of exotic compounds. Second, these lipids are atypically organized into a Gram-negative-like outer membrane (mycomembrane) in these Gram-positive bacteria, as recently revealed by CEMOVIS, and this mycomembrane also contains pore-forming proteins. Third, the mycolic acids esterified a holistic heteropolysaccharide (arabinogalacan), which in turn is linked to the peptidoglycan to form the cell wall skeleton (CWS). In slow-growing pathogenic mycobacterial species, this giant structure is surrounded by a capsular layer composed mainly of polysaccharides, primarily a glycogen-like glucan. The CWS is separated from the plasma membrane by a periplasmic space. A challenging research avenue for the next decade comprises the identification of the components of the uptake and secretion machineries and the isolation and biochemical characterization of the mycomembrane.

Isolation of Lautropia mirabilis from oral cavities of human immunodeficiency virus-infected children.

Lautropia mirabilis, a pleomorphic, motile, gram-negative coccus, has been isolated from the oral cavities of 32 of 60 (53.3%) children infected with human immunodeficiency virus (HIV) and 3 of 25 (12.0%) HIV-uninfected controls; the association of L. mirabilis isolation with HIV infection is significant (P < 0.001). All children in the study, both HIV-infected children and controls, were born to HIV-infected mothers. The presence of this bacterium was not associated with clinical disease in these children. The HIV-infected children with L. mirabilis did not differ from the HIV-infected children without L. mirabilis in immunological status, clinical status, or systemic medications. The role of HIV infection itself or concomitant factors in the establishment of L. mirabilis in the oral cavity remains to be elucidated.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.