Acquired antibiotic resistance in lactic acid bacteria from food

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. -type replicating plasmids of the pAM1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29'871 bp resistance plasmid detected in Lactococcus lacti s subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabo lic traits to generate food-grade vectors.     

Über die Zerstörung der cytoplasmatischen Membran vonSaccharomyces cerevisiae durch methylisothiocyanathaltige Bodenentseuchungsmittel

Zusammenfassung 1. Das methylisothiocyanathaltige Bodenentseuchungsmittel Trapex zerstört in wässriger Suspension (0,1–10,0%) die cytoplasmatische Membran vonSaccharomyces cerevisiae, und zwar vorwiegend durch seinen Gehalt an Methylsenföl, weniger durch das Lösungsmittel Xylol.2. Dies läßt sich aus der starken Erhöhung der Wasserstoffperoxydzersetzung und Pyrogalloloxydation in vivo nach Trapexbehandlung schließen, da Reinenzyme (in vitro) nicht aktiviert werden.3. Als direkter Beweis für die Zerstörung der Membran wird das konzentrations-und zeitabhängige Auftreten freier Aminosäuren, UV-absorbierender (260 nm) Substanzen (Purin und Pyrimidinderivate) und Folin-Ciocalteupositiver Verbindungen (Peptide bzw. Tyrosin und Tryptophan) im Filtrat gewertet.4. Nach Hitze-, Trapex- und Xyloleinwirkung sind die gleichen Aminosäuren im gleichen Verhältnis zueinander dünnschichtchromatographisch im Filtrat nachweisbar.5. Das methylsenfölhaltige Nematozid WN 12 scheint wenigstens teilweise ebenfalls durch Zerstörung der Membran zu wirken (Steigerung der H₂O₂-Zersetzung in vivo, nicht in vitro).6. Es wird weniger ein Angreifen des Methylisothiocyanats an SH-Gruppen als an den Phospholipiden der Zellmembran diskutiert.

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Summary 1. The methylisothiocyanate (MITC)-containing soil fumigant TRAPEX destroys in aqueous suspensions (0.01–10%) the cytoplasmic membrane ofSaccharomyces cerevisiae (bakers' yeast). This effect is caused mainly by the active agent MITC, in a smaller degree by the solvent xylene.2. Evidence for it is the observed activation of H₂O₂-destruction and pyrogallol oxidation in yeast suspensions after addition of Trapex (Fig. 1, 2, and 6). Commercial available enzymes (catalase, EC No. 1.11.1.6, and peroxidase, EC No. 1.11.1.7) and cell free extracts of yeast don't get activated (Fig. 3, 4, and 6).3. The appearance of UV-absorbing substances (260 nm) and protein-like material in the cell free filtrate after application of Trapex is believed to be direct evidence for a damaged membrane (Fig. 7 and 8).4. The patterns of amino-acids, which are found in the filtrate after action of Trapex, xylene, and heating, were shown by thin-layer chromatography to be identical or at least very similar (Fig. 9).5. The MITC-containing nematicide WN12 is suggested to act like Trapex.6. The point of attack of MITC in the cytoplasmic membrane may be phospholipids and not SH-groups.

     

Differential fluorescence labelling with 5-dimethyl-aminonaphthalene-1-sulfonyl chloride of intact cells and isolated membranes in Salmonella typhimurium and Acholeplasma laidlawii

For fluorescence labelling intact cells and isolated cell envelopes (membranes) from Salmonella typhimurium and Acholeplasma laidlawii were treated with mixed dansylchloride-lecithin-cholesterol vesicles. This kind of dansylation, which has been supposed to be specific for cell surface proteins, produced fluorescent protein pattern after SDS-polyacrylamide gel electrophoresis only when isolated envelopes were dansylated. Acid hydrolysis of fluorescent cell envelopes of Salmonella typhimurium yielded O-dansyltryosine and epsilon-N-dansyl-lysine besides the free sulfonic acid and unidentified compounds. However, no fluorescent proteins were detectable in cell envelopes isolated from dansylated intact bacteria from Salmonella typhimurium. In accord Acholeplasma laidlawii showed only fluorescence from proteins with a molecular weight higher than 100000.     

Bacteriophage receptors of Lactococcus lactis subsp. 'diacetylactis' F7/2 and Lactococcus lactis subsp. cremoris Wg2-1

Bacteriophage P008 revealed irreversible and uniform adsorption to cell walls of L. lactis subsp. 'diacetylactis' F7/2, whereas phage P127 adsorbed reversibly to a limited number of receptor sites on cell walls of L. lactis subsp. cremoris Wg2-1. Neither extraction of lipids, cell wall- and membrane-teichoic acids nor enzymatic degradation of proteins altered the binding efficiencies of both cell wall fractions. However, phage binding was inhibited, when cell walls were subjected to lysozyme, metaperiodate, or acid treatments. This reflects that a carbohydrate component embedded in the peptidoglycan matrix is part of the phage receptors of strains F7/2 and Wg2-1.     

Effect of polymyxin B on the structure and the stability of lipid layers

Summary Polymyxin B (PX) does not penetrate phospholipid monolayers and bilayers at low field strength across the lipid layers. The degree of penetration of PX is evaluated from its effect on the capacitance of the monolayers and on the conductance of the bilayers. PX added to one side of a bilayer causes its destabilization, it also enhances destabilization of lipid monolayers at positive electric fields across the surface layer in the direction of the adsorbed PX. PX lowers very little the fluorescence polarization of 1,6-diphenyl 1,3,5 hexatriene embedded in phospholipid vesicles. It is suggested that the penetration mechanism of PX into gram-negative bacteria is based on transient local breakdown of the plasma membrane.     

Purification and characterization of the lytic activity induced by the prolate-headed bacteriophage P001 in Lactococcus lactis

The lytic activity induced by the lactococcal bacteriophage P001 was isolated from phage lysates of Lactococcus lactis by a four-step purification procedure. Two proteins lytic for L. lactis were identified with molecular weights of 28 kDA and 8 kDa, respectively. The N-terminal amino acid sequences of the two proteins were determined and degenerated oligonucleotide probes corresponding to these sequences were synthesized. DNA hybridization experiments with phage P001-DNA and lactococcal DNA revealed that both proteins were apparently encoded by a single lysin gene located on the phage P001 genome. This was confirmed by alignment of the determined N-terminal amino acid sequences with nucleotide sequences which were deduced from cloned Lactococcus bacteriophage lysin genes.