Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference 2013.

Bacterial infections are very common and represent one of the most important reasons of progression of liver failure, development of liver-related complications, and mortality in patients with cirrhosis. In fact, bacterial infections may be a triggering factor for the occurrence of gastrointestinal bleeding, hypervolemic hyponatremia, hepatic encephalopathy, kidney failure, and development of acute-on-chronic liver failure. Moreover, infections are a very common cause of repeated hospitalizations, impaired health-related quality of life, and increased healthcare costs in cirrhosis. Bacterial infections develop as a consequence of immune dysfunction that occurs progressively during the course of cirrhosis. In a significant proportion of patients, infections are caused by gram-negative bacteria from intestinal origin, yet gram-positive bacteria are a frequent cause of infection, particularly in hospitalized patients. In recent years, infections caused by multidrug-resistant bacteria are becoming an important clinical problem in many countries. The reduction of the negative clinical impact of infections in patients with cirrhosis may be achieved by a combination of prophylactic measures, such as administration of antibiotics, to reduce the occurrence of infections in high-risk groups together with early identification and management of infection once it has developed. Investigation on the mechanisms of altered gut microflora, translocation of bacteria, and immune dysfunction may help develop more effective and safe methods of prevention compared to those that are currently available. Moreover, research on biomarkers of early infection may be useful in early diagnosis and treatment of infections. The current manuscript reports an in-depth review and a position statement on bacterial infections in cirrhosis.

Response of gram-positive bacteria to copper stress

The Gram-positive bacteria Enterococcus hirae, Lactococcus lactis, and Bacillus subtilis have received wide attention in the study of copper homeostasis. Consequently, copper extrusion by ATPases, gene regulation by copper, and intracellular copper chaperoning are understood in some detail. This has provided profound insight into basic principles of how organisms handle copper. It also emerged that many bacterial species may not require copper for life, making copper homeostatic systems pure defense mechanisms. Structural work on copper homeostatic proteins has given insight into copper coordination and bonding and has started to give molecular insight into copper handling in biological systems. Finally, recent biochemical work has shed new light on the mechanism of copper toxicity, which may not primarily be mediated by reactive oxygen radicals.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Bacterial infections in horses: a retrospective study at the University Equine Clinic of Bern

Bacterial infections present a major challenge in equine medicine. Therapy should be based on bacteriological diagnosis to successfully minimize the increasing number of infections caused by multidrug-resistant bacteria. The present study is a retrospective analysis of bacteriological results from purulent infections in horses admitted at the University Equine Clinic of Bern from 2004 to 2008. From 378 samples analyzed, 557 isolates were identified, of which Staphylococcus aureus, Streptococcus equi subsp. zooepidemicus and coliforms were the most common. Special attention was paid to infections with methicillin-resistant S. aureus (MRSA) ST398 and a non-MRSA, multidrug-resistant S. aureus clone ST1 (BERN100). Screening of newly-admitted horses showed that 2.2 % were carriers of MRSA. Consequent hygiene measures taken at the Clinic helped to overcome a MRSA outbreak and decrease the number of MRSA infections.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Antimicrobial susceptibility of gram-positive udder pathogens from bovine mastitis milk in Switzerland

We evaluated the susceptibility of the gram-positive mastitis pathogens S. aureus, Str. uberis, Str. dysgalactiae, E. faecalis and L. garviae to antibiotics that are of epidemiological interest or are critically important for mastitis therapy and human medicine. Penicillin resistance was found to be most frequent in S. aureus, and nearly 5 % of the Str. uberis strains displayed a decreased susceptibility to this antibiotic. Resistance to aminoglycosides and macrolides was also detected in the strains tested. The detection of methicillin-resistant S. aureus (MRSA) and of a ciprofloxacin-resistant Str. dysgalactiae isolate corroborated the emergence of mastitis pathogens resistant to critically important antibiotics and underscores the importance of susceptibility testing prior to antibiotic therapy. The monitoring of antibiotic susceptibility patterns and antibiogram analyses are strongly recommended for targeted antimicrobial treatment and to avoid the unnecessary use of the latest generation of antibiotics.

Conjugal Transfer of the Tn916-like Transposon TnFO1 from Enterococcus faecalis Isolated from Cheese to Other Gram-positive Bacteria

A Tn916-like transposon (TnFO1) was found in the multiple antibiotic resistant Enterococcus faecalis strain FO1 isolated from a raw milk cheese. In this strain, the tetracycline determinant was localized by DNA-DNA hybridization with a tetM nucleotide probe on the chromosome and on a 30-kb plasmid. The transposon TnFO1 was identified and characterized by DNA-DNA hybridization experiments with the five internal HincII fragments of Tn916. The tetracycline resistance determinant was identified by its complete nucleotide sequence as TetM. Transposon TnFO1 was also detected in its circular form by DNA-DNA hybridization and PCR amplification. Both ends including the joining region of the closed circular transposon TnFO1 were sequenced. TnFO1 could be transferred by conjugation from Enterococcus faecalis into Enterococcus faecalis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Listeria innocua, Leuconostoc mesenteroides and Staphylococcus aureus, and from Lactococcus lactis subsp. lactis biovar. diacetylactis into Listeria innocua. Pulsed-field electrophoresis of genomic DNA from E. faecalis FO1 transconjugants showed that transposon TnFO1 integrated at different sites.

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

Bacterial decontamination of surgical wounds treated with Lavasept

In a prospective randomized controlled double-blind study in 50 acutely injured patients, bacterially contaminated type 2-4 soft tissue wounds were treated with moist dressings of 0.2% Lavasept (fractionated polyhexamethylenbiguanide and macrogolum 4000) solution (n=28) in comparison with Ringer solution (n=22). Standardized swabs were taken on days 0, 2, 8 and 15 and investigated for microorganisms. For a quantitative evaluation, the number of colony forming units (CFU) was determined by a serial dilution technique. The tissue compatibility and anti-inflammatory effect were rated on a scale of 0 (=bad) to 3 (=very good). The most frequently found microorganism was Staphylococcus aureus, which was isolated from 13 wounds. Use of Lavasept led to a faster and significant reduction in microorganisms on the wound surfaces. The number of CFU per wound remained constant or decreased, in contrast to the wounds treated with Ringer solution. This was true for both Gram-positive and Gram-negative bacteria. There was no evidence of impaired wound healing in either group. The anti-inflammatory effect and the tissue compatibility of Lavasept were rated significantly better than that of Ringer solution. It is concluded that Lavasept combines antiseptic action with good tissue compatibility.