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.

Purification and some properties of ATP-dependent deoxyribonuclease of Caulobacter crescentus.

An ATP-dependent deoxyribonuclease has been partially purified from extracts of Caulobacter crescentus cells in a procedure involving ion-exchange and affinity chromatography. The enzyme was purified approximately 350-fold and was free of contaminating nucleolytic and ATPase activity. The nuclease hydrolyzes linear, double-stranded DNA with subsequent release of short oligonucleotides, mostly from one to four bases in length. The release of nucleotides is accompanied by hydrolysis of ATP, 7.6 nmol ATP being consumed for each nmol of acid-soluble products of DNA degradation. The enzyme shows an absolute requirement for divalent cations and in most active at pH 7.6 to 8.8. The molecular weight of the nuclease, estimated by gel filtration and sucrose density gradient centrifugation, is 280 000.

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.

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.

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.

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.

[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.

Short communication: ATP-dependent deoxyribonuclease activity in segregated cell types of Caulobacter crescentus.

Partially purified bacterial extracts were prepared from segregated stalked and swarmer bacteria of Caulobacter crescentus and assayed for ATP-dependent deoxyribonuclease activity. This activity was found to be very low in the swarmer bacteria compared with a pure population of stalked cells or an asynchronous population of C. crescentus.

Murein hydrolases of Caulobacter crescentus.

Caulobacter crescentus was found to exhibit a similar autolytic response to a variety of factors affecting the structure of the cell envelope and interfering with murein synthesis as several other species of bacteria. Autolysis was accompanied by the hydrolysis of murein with the release of soluble degradation products. Several murein hydrolases with different bond specificity were found and except for the absence of DD-carboxypeptidase and LD-carboxypeptidase activities the make-up of these enzymes resembled that of the well studied bacterium Escherichia coli.

[Spleno-gonadal fusion. Apropos of 3 case reports]. / Fusion spléno-gonadique. A propos de 3 observations.

Splenogonadal fusion is a very rare congenital anomaly, usually diagnosed during operation for other surgical conditions as inguinal during operation for other surgical conditions as inguinal hernia, testicular tumor, cryptorchism or epididymitis and discontinuous. In first form exists a link of splenic and connective-fibrous tissue between spleen and testicle. This string runs above the bowels, then through an inguinal canal and joins the testicle or surrounds the gonad. Surgical treatment consists of cutting of the cord from spleen and separating from albugineals coat of testicle. In discontinuous form numerous tubercles of splenic tissue situated near gonad are confirmed. Three own cases are presented. First case concerns a 4 year-old boy with diagnosis: tumor of testicle and left inguinal hernia. Splenic tissue surrounded testicle and separated it from epididymis. An attempt of separation of these masses from the testicle was unsuccessful and hemicastration was performed and removed 17 cm long cord attached to the spleen. There was no union between testicle and epididymis on micro- and macroscopic examination. Second case refers to a 8 year-old boy with left-sides cryptorchidism. During an operation 7 cm long string like rosary was removed. Last case: 12 year-old boy was treated with diagnosis of tumor of spermatic cord and suspicion of spleno-gonadal fusion. A 15 cm long string connecting upper pole of testicle and spleen was amputated. The embryology, the diagnosis and the treatment are discussed in both cases.

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.

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.

Isolation of mutants of Caulobacter crescentus deficient in ATP-dependent deoxyribonuclease activity.

Caulobacter crescentus mutants sensitive to UV radiation, mitomycin C and methyl methane-sulfonate were isolated and tested for ATP-dependent deoxyribonuclease activity. Two mutants were identified of which one had less than 5 per cent of the wild-type level of the nuclease activity. This mutant in cross with another auxotrophic partner gave highly reproducible two-fold reduction in number of recombinants compared to a control cross. The suggested causes for reduced recombination frequency when one of the partners has residual ATP-dependent deoxyribonuclease activity are discussed.

Autolysis of Caulobacter crescentus grown in the presence of glycine.

Caulobacter crescentus was grown in complex medium supplemented with low (0.05%) concentration of glycine, a component of the murein peptide side chains of this bacterium. Murein synthesized in the presence of glycine was poorly crosslinked and the rate of its synthesis was slowed down compared to the control cells. The glycine-grown cells were considerably more sensitive to the chelating agent EDTA and Tris buffer than the control cells and also lysed faster when incubated with beta-lactam antibiotics. No changes in phospholipid composition in the presence of glycine were observed and the outer membrane protein composition of the glycine-grown cells was altered only in the amount of 130 000 protein which forms the surface array of C. crescentus. The effects of glycine can thus be tentatively put down to the reduced crosslinkage of murein synthesized in its presence.

Characterization of the cell wall murein of Thiobacillus versutus.

Amino acid analysis of pure murein isolated from cells of Thiobacillus versutus grown in complex medium revealed the typical constituents of most mureins from gram-negative cells, i.e. muramic acid, glucosamine, glutamic acid, alanine and diaminopimelic acid in molecular ratio of 0.58: 0.79: 1.0: 1.76:1.07, respectively. The presence of glycine and leucine was also demonstrated (0.20 and 0.08 compared to glutamic acid). Glycine was also present in the murein of cells grown in chemically defined synthetic medium. The crosslinkage of T. versutus murein was approximately 36% --much higher than for most other gram-negative species. High pressure liquid chromatography analysis of muropeptide composition following muramidase digestion of T. versutus murein revealed essentially the same pattern as for Escherichia coli under similar conditions of digestion and separation with, however, some differences in the minor peaks.

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.

Mutants of Caulobacter crescentus resistant to beta-lactam antibiotics.

A number of mutants of Caulobacter crescentus CB15 resistant ot ampicillin were isolated. The mutants differred in their resistance to several beta-lactam antibiotics. No differences in composition of the penicillin-binding proteins of the mutants compared to the parental strain, or in the affinity of these proteins to penicillin or ampicillin were found. The mutants were found to differ from the parent and also in many cases from each other in outer membrane protein composition.

Characterization of a Helical Morphological Mutant of Caulobacter crescentus.

Screening of poorly growing colonies following EMS treatment of Caulobacter crescentus culture revealed several mutants with altered cell morphology. One of these (CB15-UW.5) grew as elongated, helically twisted cells which were more susceptible to autolysis than the wild type. The mutant cells were found to have higher transglycosylase activity, this being reflected by shorter chain length of their murein. A second difference between the murein of the mutant and the parent was lower trimer content in the former, although in both cases the degree of crosslinkage was similar.