The intrinsic cephalosporin resistome of Listeria monocytogenes in the context of stress response, gene regulation, pathogenesis and therapeutics.

Intrinsic resistance to antibiotics is a serious therapeutic problem in the case of many bacterial species. The Gram-positive human pathogen Listeria monocytogenes is intrinsically resistant to broad spectrum cephalosporin antibiotics, which are commonly used in therapy of bacterial infections. Besides three penicillin-binding proteins the intrinsic cephalosporin resistome of L. monocytogenes includes multidrug resistance transporter transporters, proteins involved in peptidoglycan biosynthesis and modification, cell envelope proteins with structural or general detoxification function, cytoplasmic proteins with unknown function and regulatory proteins. Analysis of the regulation of the expression of genes involved in the intrinsic resistance of L. monocytogenes to cephalosporins highlights the high complexity of control of the intrinsic resistance phenotype. The regulation of the transcription of the intrinsic resistome determinants involves the activity of eight regulators, namely LisR, CesR, LiaR, VirR, σ(B) , σ(H) , σ(L) and PrfA, of which the most prominent role play LisR, CesR and σ(B) . Furthermore, the vast majority of the intrinsic resistome determinants contribute to the tolerance of different stress conditions and virulence. A study indicates that O-acetyltransferase OatA is the most promising candidate for co-drug development since an agent targeting OatA should sensitize L. monocytogenes to certain antibiotics, therefore improving the efficacy of listeriosis treatment as well as food preservation measures.

Listeria monocytogenes protein p60 affects hemolytic activity and uptake of bacteria by macrophages.

Bacillus subtilis strains expressing listeriolysin O (LLO) and simultaneously LLO and p60 protein were constructed. The effect of p60 protein on hemolytic activity and on the invasion of professional phagocytes was demonstrated in the absence of other virulence factors of L. monocytogenes. The hemolytic activity of LLO in the presence of p60 protein decreased which indicates that p60 promoted adhesion and subsequent invasion of professional phagocytes.

Autolysis of Listeria monocytogenes.

Physiological conditions that could provide maximal rates of autolysis of Listeria monocytogenes were examined. L. monocytogenes was found to be refractory to most treatments that promote rapid autolysis in other bacteria. Best rates of autolysis were obtained after resuspending the cells in Tris-hydrochloride buffer at 37 degrees C with the pH optimum at 8.0. Autolysis was also efficiently promoted by the surfactant Triton X-100. Antibiotics that interfere with the biosynthesis of the cell wall murein (peptidoglycan) caused death of the cells without autolysis after prolonged incubation in the presence of the drug. Only nisin, which has been shown to bind in vitro to the murein precursors lipid I and lipid II brings about autolysis of L. monocytogenes cells, although with slower kinetics than in the case of Tris-HCl and Triton.

Effect of benzylpenicillin on murein biosynthesis, turnover and structure in Listeria monocytogenes cells.

The action of beta-lactam antibiotics such as ampicillin and benzylpenicillin on cells of Listeria monocytogenes appears to be bacteriostatic. However, after approximately two hours in the presence of 10 x MIC of benzylpenicillin the cells begin to rapidly lose viability without undergoing lysis. In this report we present the results of studies on the biosynthesis of murein in L. monocytogenes cells during the first 120 min of their exposure to benzylpenicillin as measured by the continuous and pulse incorporation of the precursor N-acetyl-D-[1-3H]glucosamine. The turnover of the murein sacculus in the presence of penicillin as well as the lack of discernible changes in the molecular structure of the murein synthesised in the presence of benzylpenicillin is also discussed.

Structure of cell envelope components of the primary and secondary forms of Xenorhabdus luminescens.

Muropeptide analysis of muramidase-digested murein (peptidoglycan) did not reveal any differences between the primary and secondary forms of Xenorhabdus luminescens. Similarly, no significant differences were found in the overall protein composition of the cytoplasmic and outer membranes of both forms.

Relationship between phase variation in colony morphology, intrastrain variation in cell wall physiology, and nasopharyngeal colonization by Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes phase variation in colony morphology, which has been implicated as a factor in the pathogenesis of pneumococcal disease. Phenotypic differences between opaque and transparent colony forms correlate with differences in rates of autolysis. This study examined whether differences in autolysis are caused by differences in expression of the major amidase, LytA, or the structure of its peptidoglycan substrate. No significant difference was detected by high-pressure liquid chromatography analysis of stem peptides released after treatment of purified peptidoglycan with amidase. Differences in the rate of digestion of purified cell walls, furthermore, did not correlate with susceptibility to autolysis. Lower levels of autolysis in opaque variants, however, was associated with decreased levels of immunodetectable LytA on colony immunoblots and Western blots (immunoblots). Diminished cell-surface-associated LytA in opaque variants was also demonstrated by whole-cell inhibition enzyme-linked immunosorbent assay. Since transparent variants have been shown both to colonize the nasopharynx more efficiently in an animal model and to express more surface-exposed LytA, it was determined whether LytA contributes to colonization in a neonatal rat model of pneumococcal carriage. Defined mutants in the lytA gene were used to show that there was no significant contribution by LytA to nasopharyngeal colonization in this model. Although the expression of LytA was shown to undergo phase variation in association with colony morphology, lytA mutants are still capable of phenotypic variation in colony morphology, which suggests that other factors are responsible for intrastrain differences which affect colonization.

Failure to trigger the autolytic enzymes in minicells of Escherichia coli.

Minicells from Escherichia coli P678-54 are refractory towards procedures known to induce bacteriolysis of DNA-containing E. coli cells. Although still engaged in murein synthesis, minicells could not be lysed by penicillin G. Likewise, endogenous overproduction of the cloned soluble lytic transglycosylase, the predominant murein hydrolytic activity in E. coli, failed to lyse minicells. Furthermore, induction of the phage MS2 lysis protein, a hydrophobic protein assumed to trigger the autolytic system of the host, did not result in bacteriolysis. It is concluded that the murein hydrolases present in minicells are under a tight cellular control.

[Problems encountered in identification of penicillin binding proteins with pneumococcus used as an example]. / Problemy zwiazane z identyfikacja bakteryjnych bialek wiazacach penicyline na przykladzie dwoinki zapalenia pluc.

Whole cells or isolated membranes of Streptococcus pneumoniae were treated with labelled benzyl penicillin and the penicillin binding proteins (PBPs) were visualized by fluorography after SDS-PAGE electrophoresis. The PBP profiles obtained for strains sensitive and resistant to penicillin strongly differed depending not only on the concentrations of acrylamide and bis-acrylamide used in the separating gel but also on the batch used (different manufacturers). The latter was also true for sodium dodecyl sulfate.

N-unsubstituted glucosamine residues and other modifications in murein of the obligatory chemolithotroph Thiobacillus neapolitanus.

Purified murein from Thiobacillus neapolitanus was poorly digested by lysozyme. It's sensitivity to the enzyme greatly increased after N-acetylation. The murein was found to contain 30 to 35% glucosamine residues lacking N-acetyl groups. It also contained phosphomuramic acid. Further modifications included amidation of diaminopimelic acid in the peptide side chains and a low alanine content. None of these modifications were found in the murein of another sulphur bacterium, Thiobacillus versutus.

Changes in murein composition during the cell cycle of Caulobacter crescentus.

The amino acid and muropeptide compositions of murein (peptidoglycan) isolated from populations of Caulobacter crescentus predominantly composed of swarmer or stalked cells were determined and compared with the structure of murein sacculi obtained from a population of unsegregated cells. It appears that in spite of vast morphological alterations in the course of the cell cycle, the murein composition of the various cell types is not markedly different.

Novel plasmids in clinical strains of Streptococcus pneumoniae.

Small plasmids were found in two clinical isolates of Streptococcus pneumoniae from Spain (strains 671 and 678) and in one strain (SpR) isolated in Germany. All three strains contained one plasmid (2.75 to 3.1 kb) which is related to the only previously described pneumococcal plasmid, pDP1. Strains 678 and SpR carried a second plasmid of 2.6 kb and 2.7 kb, respectively. These two plasmids hybridized neither with each other, nor with pDP1, demonstrating that they represent new types of plasmids not having been found in pneumococci before.

The murein of Thiobacillus neapolitanus.

Amino acid analysis of pure murein isolated from cells of T. neapolitanus revealed the typical constituents of most mureins form Gram-negative bacteria. i.e. glutamic acid, alanine and diaminopimelic acid, but the molecular ratio ot these was unusual, being approximately 1: 1: 1. The reduced amount of alanine was explained by the absence of monomers containing tetrapeptide side chains, as revealed by h. p. 1. c. analysis, [(3)H]glutamic acid, [(3)H]diaminopimelic acid and [(3)H]N-acetylglucosamine were incorporated into the murein and allowed to determine the degree of its crosslinkage (28%) and the occurrence of turnover.

Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles.

The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline. The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution. When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site. Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein. It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.

Meningitis due to penicillin-resistant Streptococcus pneumoniae in adults.

A 33-year-old woman with quiescent systemic lupus erythematosus developed meningitis due to a penicillin-resistant strain of Streptococcus pneumoniae. After an initial failure of penicillin therapy, the patient responded to cefotaxime. The meningitis was complicated by total neurosensory hearing loss. Howell-Jolly bodies noted on peripheral blood smear led to a radionuclide spleen scan, which documented functional asplenia. This woman is the second patient in the United States with meningitis caused by a strain of S. pneumoniae moderately resistant to penicillin G (minimal inhibitory concentration, 1.0 microgram/mL). The resistant isolate displayed resistance to penicillin that was not of enzymatic origin but rather was due to an alteration of penicillin-binding proteins. This experience illustrates the importance of testing the in vitro susceptibility of the pneumococcus to penicillin G.

Two bactericidal targets for penicillin in pneumococci: autolysis-dependent and autolysis-independent killing mechanisms.

It has been assumed that penicillin (and also other cell wall inhibitors) kill pneumococci predominantly by triggering their major autolytic enzyme (an N-acetylmuramoyl-L-alanine amidase; referred to as amidase), resulting in massive cell wall degradation. Three types of experiments suggest that only part of this killing is due to cell lysis by amidase. (i) Suppression of penicillin-induced lysis by specific inhibitors of amidase protected pneumococci only marginally from killing in spite of prolonged exposure to concentrations of penicillin that were 10x, 20x, or 100x greater than the MIC. (ii) Mutants from which the amidase was completely eliminated by plasmid insertion or deletion (Lyt-) were still killed, albeit at a slower rate than the wild-type Lyt+ strains (3 to 4 log units instead of 4 to 5 log units per 6 h, i.e., about 1 log unit slower than the wild type; P less than 0.001). (iii) A new mutation (cid), which was not related to the amidase gene, further reduced killing of mutants lacking amidase to 1 log unit per 6 h (Lyt- Cid- phenotype). Reintroduction of the amidase gene into Lyt- Cid- cells partially restored penicillin-induced lysis but increased only slightly the rate of killing (from 1 log unit per 6 h in Lyt- Cid- cells to 2 log units per 6 h in Lyt+ Cid- cells). We conclude that penicillin kills pneumococci by two distinct mechanisms: one that involves the triggering of the amidase (about 1 log unit of killing per 6 h) and another, amidase-independent mechanism that is responsible for 3 to 4 log units of killing per 6 h. Triggering of the amidase activity in situ in growing bacteria was significantly reduced in Lyt+ Cid- cells, indicating that there is some regulatory interaction between the cid gene product and the amidase.

Role of autolysins in the activities of imipenem and CGP 31608, a novel penem, against slowly growing bacteria.

The carbapenem imipenem and the penem CGP 31608 demonstrated unusually good bactericidal activity against slowly growing bacteria. In contrast to that of penicillin, the rate of killing was independent of growth rate. In logarithmically growing cells, a decrease in growth rate was paralleled by a decrease in the relative activity of only one of four autolysins measured (membrane-bound endopeptidase), suggesting that autolysis induced by penicillin G may be rate limited by this enzyme. Imipenem, on the other hand, appeared to trigger different autolysins in Escherichia coli, as evidenced by differences in the structure of peptidoglycan after imipenem- versus penicillin-induced autolysis.

Variation in penicillin-binding protein patterns of penicillin-resistant clinical isolates of pneumococci.

A large number of pneumococcal isolates (over 80 strains) from a variety of geographic locales and representing a spectrum of resistance levels from a penicillin MIC of 0.003 microgram/ml up to an MIC of 16 micrograms/ml were analyzed for their penicillin-binding protein (PBP) patterns. With a few exceptions, the great majority of strains with penicillin MICs up to about 0.05 microgram/ml contained the same set of five PBPs with molecular sizes typical of those of susceptible pneumococci. In strains with penicillin MICs of about 0.1 microgram/ml and up, virtually all isolates showed two common features: (i) all isolates showed loss of PBP 1A (98 kilodaltons) with or without a parallel appearance of a "new" PBP that ranged in molecular size between 96 and 97 kilodaltons; and (ii) in strains with penicillin MICs of 0.5 microgram/ml or more, PBP 2B could not be detected on the fluorograms even with very high concentrations of radioactive penicillin. Beyond these two common features, resistant strains with similar penicillin MICs showed a surprising variety of PBP profiles (i.e., in the number and molecular sizes of PBPs), each characteristic of a given isolate. We suggest that in pneumococci remodeling of critical PBPs in more than one way may result in comparable levels of penicillin resistance.