Martti Vaara

Polycations as outer membrane-disorganizing agents.

The outer membrane-disorganizing effect of a short (10-min) treatment with polycationic agents was studied with smooth Salmonella typhimurium used as a test organism. The polycationic agents were the protamine salmine, a lysine polymer with 20 lysine residues (lysine20), and the deacylated polymyxin B derivative polymyxin B nonapeptide. Two different types of outer membrane-disorganizing were found. Protamine and lysine20 released 20 to 30% of the lipopolysaccharide from the outer membrane and sensitized the bacteria to the anionic detergent sodium dodecyl sulfate but did not (under these conditions) make the bacteria permeable to the hydrophobic probes fusidic acid and actinomycin D. In contrast, polymyxin B nonapeptide did not release lipopolysaccharide or sensitize the bacteria to sodium dodecyl sulfate but made the outer membrane permeable to the hydrophobic probes. None of the agents was bactericidal under the conditions used or caused any leakage of periplasmic beta-lactamase. Polymyxin B was used as a reference and showed characteristic outer membrane-disorganizing action. In thin-section electron microscopy, polymyxin B nonapeptide caused the appearance of long, narrow, finger-like projections on the outer membrane. Protamine and lysine20 caused a distinctly wrinkled appearance of the outer membrane but no projections. Images

Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: an observational study

BACKGROUND New DNA-based microarray platforms enable rapid detection and species identification of many pathogens, including bacteria. We assessed the sensitivity, specificity, and turnaround time of a new molecular sepsis assay. METHODS 2107 positive blood-culture samples of 3318 blood samples from patients with clinically suspected sepsis were investigated for bacterial species by both conventional culture and Prove-it sepsis assay (Mobidiag, Helsinki, Finland) in two centres (UK and Finland). The assay is a novel PCR and microarray method that is based on amplification and detection of gyrB, parE, and mecA genes of 50 bacterial species. Operators of the test assay were not aware of culture results. We calculated sensitivity, specificity, and turnaround time according to Clinical and Laboratory Standards Institute recommendations. FINDINGS 1807 of 2107 (86%) positive blood-culture samples included a pathogen covered by the assay. The assay had a clinical sensitivity of 94.7% (95% CI 93.6-95.7) and a specificity of 98.8% (98.1-99.2), and 100% for both measures for meticillin-resistant Staphylococcus aureus bacteraemia. The assay was a mean 18 h faster than was the conventional culture-based method, which takes an additional 1-2 working days. 34 of 3284 (1.0%) samples were excluded because of technical and operator errors. INTERPRETATION Definitive identification of bacterial species with this microarray platform was highly sensitive, specific, and faster than was the gold-standard culture-based method. This assay could enable fast and earlier evidence-based management for clinical sepsis.

Polycations sensitize enteric bacteria to antibiotics.

Polymyxin B nonapeptide, a polymyxin B derivative which lacks the fatty acyl part and the bactericidal activity of polymyxin, was shown to sensitize smooth encapsulated Escherichia coli (O18:K1) and smooth Salmonella typhimurium to hydrophobic antibiotics (novobiocin, fusidic acid, erythromycin, clindamycin, nafcillin, and cloxacillin). The polymyxin B nonapeptide-treated bacteria were as sensitive to these antibiotics as are deep rough mutants. A lysine polymer with 20 lysine residues (lysine 20) had a largely similar effect. Larger lysine polymers and the protamine salmine were bactericidal but, at sublethal concentrations, sensitized the strains to the antibiotics mentioned above, whereas lysine4, streptomycin, cytochrome c, lysozyme, and the polyamines cadaverine, spermidine, and spermine had neither bactericidal nor sensitizing activity.

Lipopolysaccharides of polymyxin B‐resistant mutants of Escherichia coii are extensively substituted by 2‐aminoethyl pyrophosphate and contain aminoarabinose in lipid A

Lipopolysaccharides (LPS) of two polymyxin‐resistant (pmr) mutants and the corresponding parent strain of Escherichia Coli were chemically analysed for composition and subjected to 31P‐NMR (nuclear magnetic resonance) for assessment of phosphate substitution. Whereas the saccharide portions, fatty acids, and phosphate contents were similar in wild‐type and pmr LPS, the latter contained two‐ to threefold higher amounts of 2‐aminoethanol. The pmr LPS also contained 4‐amino‐4‐deoxy‐l‐arabinopyranose (l‐Arap4N), which is normally not a component of E. coli LPS. This aminopentose has been assigned to be linked to the 4′‐phosphate of lipid A. Comparative 31P‐NMR analysis of the de‐O‐acylated LPS of the wild‐type and pmr strains revealed that phosphate groups of the pmr LPS were mainly (71‐79%) diphosphate diesters, which accounted for only 20% in the wild‐type LPS. Diphosphate monoesters were virtually nonexistent in the pmr LPS, whereas they accounted for 42% of all phosphates in wild‐type LPS. In the lipid A of the pmr strains, the 4′‐phosphate was to a significant degree (35%) substituted by l‐Arap4N, whereas in the wild‐type LPS the l‐ArapN was absent. In the pmr lipid A1 2‐aminoethanol was completely substituting the glycosidic pyrophosphate but not the glycosidic monophosphate, forming a diphosphate diester linkage at this position in 40% of lipid A molecules. In the wild‐type LPS the glycosidic position of lipid A carried mostly unsubstituted monophosphate and pyrophosphate. Thus the polymyxin resistance was shown to be associated, along with the esterification of the lipid A 4′‐monophosphate by aminoarabinose, with extensive esterification of diphosphates in LPS by 2‐aminoethanol.

Increased substitution of phosphate groups in lipopolysaccharides and lipid A of the polymyxin‐resistant pmrA mutants of Salmonella typhimurium: a 31P‐NMR study

De‐O‐acylated lipopolysaccharides (LPS) of three polymyxin‐resistant Salmonella typhimurium pmrA mutants and their parent strains were analysed by 31P‐NMR (nuclear magnetic resonance) in order to assess, in relation to polymyxin resistance, the types and degree of substitution of phosphates of the LPS and lipid A. in the pmrA mutant LPS phosphate diesters predominated over phosphate monoesters, whereas the latter were more abundant in the parent wild‐type LPS. The increase in the proportion of phosphate diesters was traced to both the core oligosaccharide and the lipld A part. In the latter, the ester‐linked phosphate at position 4’was to a large extent (79–88%) substituted with 4‐amino‐4‐deoxy‐l‐arabinose, whereas in the wild‐type LPS the 4′‐phosphate was mainly present as monoester. In each LPS, regardless of the pmrA mutation, the glycosidically linked phosphate of lipid A was largely unsubstituted.

Sensitization of Gram-negative bacteria to antibiotics and complement by a nontoxic oligopeptide

A major virulence factor of bacteria that cause generalized infections is their resistance to the lytic action of the complement cascade1, an important defence mechanism of the host. Invasive Gram-negative enteric bacteria, which cause about one-third of all bacteraemic infections2,3, are completely resistant to lysis by complement, even in the presence of hyperim-mune serum4–6. The same bacteria are also resistant to many antibiotics that are effective therapeutic agents against other bacteria, as the outermost surface layer (the outer membrane) of the bacteria functions as a permeability barrier7. Here we show that it is possible to sensitize such bacteria to both complement and antibiotics by using an agent that binds to the outer membrane. This agent is a nontoxic derivative of polymyxin which by itself has no bactericidal action8,9.

Neonatal Candida parapsilosis outbreak with a high case fatality rate

A Candida parapsilosis outbreak of 58 cases in a neonatal intensive care unit lasted for 55 months. Patients infected by or colonized with C. parapsilosis were mainly very low birth weight infants (birth weight < 1500 g). Their mean birth weight was 817 g and their mean gestational age was 28 weeks. Statistical analysis including logistic regression confirmed that prematurity was the main risk factor. The analysis also suggested that C. parapsilosis infection (or colonization) was associated with a poor prognosis. In infants with gestational age < 29 weeks the risk for death in C. parapsilosis-infected patients was 16-fold greater than in those with no C. parapsilosis infection. The case fatality rate of C. parapsilosis patients was higher than that of the controls (9 of 23 vs. 1 of 40; P < 0.0001). The outbreak was most likely a result of cross-infection because C. parapsilosis could be isolated only from the patients and from the hands of four nurses immediately after they had cared for a colonized patient. Cessation of the outbreak was temporally associated with long term parenteral fluconazole (6 mg/kg/day) prophylaxis.

Novel Polymyxin Derivatives Carrying Only Three Positive Charges Are Effective Antibacterial Agents

ABSTRACT The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-d-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.

New approaches in peptide antibiotics

The emergence and rapid spread of extremely multiresistant bacteria necessitates every effort to develop novel antibacterial agents. Host-derived antimicrobial peptides (AMPs) pexiganan (a magainin), omiganan (an indolicidin), and iseganan (a protegrin) have been in clinical phases for more than 10 years and provide little in combating against those bacteria. Even though some recent approaches may yield more effective and better-tolerated derivatives of host-derived AMPs, most novel derivatives suffer from weak activity, nonspecific cytotoxicity, and apparent susceptibility to proteolysis. Few have undergone any systematic toxicity and efficacy studies. Bacterial peptides and their derivatives, such as lantibiotics, the RTA-3 peptide, and novel polymyxins, as well as certain peptidomimetics, such as ceragenins, might be more useful.

The lipid A biosynthesis mutation lpxA2 of Escherichia coli results in drastic antibiotic supersusceptibility.

The conditionally lethal lpxA2 mutant of Escherichia coli, which lacks detectable UDP-N-acetylglucosamine acyltransferase activity and which produces greatly reduced amounts of lipid A after a shift to 42 degrees C (S. Galloway and C. R. H. Raetz, J. Biol. Chem. 265:6394-6402, 1990), was found to be, at conditions which promote normal growth, remarkably susceptible to a number of antibiotics. The MICs of hydrophobic antibiotics, such as rifampin, erythromycin, clindamycin, and fusidic acid, were 32- to greater than 128-fold lower for the lpxA2 strain than for the parent type strain, and those of the peptide antibiotics vancomycin and bacitracin were 32- and 256-fold lower, respectively. Futhermore, the lpxA2 strain was found to be sensitive to hypoosmotic conditions. Comparisons with the other characterized outer membrane permeability mutants, such as the heptose-deficient strains of E. coli and Salmonella typhimurium, the acrA and abs mutants of E. coli, and the ssc-1 and class SS-B mutants of S. typhimurium, indicated that the lpxA2 mutant had characteristically the most antibiotic-supersusceptible phenotype. These findings advocate the possible use of the lpxA2 strain as a tool in various fields of basic and applied bacterial research in which the impermeability of the outer membrane currently poses problems.