Michael Teuber

Veterinary use and antibiotic resistance.

Globally, an estimated 50% of all antimicrobials serve veterinary purposes. Bacteria that inevitably develop antibiotic resistance in animals comprise food-borne pathogens, opportunistic pathogens and commensal bacteria. The same antibiotic resistance genes and gene transfer mechanisms can be found in the microfloras of animals and humans. Direct contact, food and water link animal and human habitats. The accumulation of resistant bacteria by the use of antibiotics in agriculture and veterinary medicine and the spread of such bacteria via agriculture and direct contamination are documented.

Antibiotic resistance spread in food

Nutritive and therapeutic treatment of farm animals with antibiotics, amounting to half of the worlds antibiotic output, has selected for resistant bacteria that may contaminate the food produced. Antibiotic-resistant enterococci and staphylococci from animals are found in food when they survive the production processes, as in raw cured sausages and raw milk cheeses. The broad host ranges of some plasmids and the action of transposons in many bacteria allow antibiotic-resistance genes to be communicated by conjugation between different species and genera,. A multi-antibiotic resistance plasmid from a lactococcus found in cheese provides a historical record of such events.

Species and Type Phages of Lactococcal Bacteriophages

Lactococcal phages are classified according to morphology and DNA homology. Phages are differentiated into 12 phage species, and type phages of each species are proposed. Members and possible members of each species are named. Available data on type phages are tabulated including morphology, DNA characteristics and phage protein bands.

Bifidobacterium lactis sp. nov, a moderately oxygen tolerant species isolated from fermented milk

Summary A selection procedure was developed to isolate Bifidobacterium strains from food and faeces which are able to grow under oxidative stress conditions. Strain UR1, an isolate from a French yoghurt, was able to grow at elevated oxygen concentrations above 5% oxygen in liquid media containing 0.3 mM paraquat (methylviologen). In fermenter cultures, strain UR1 produced considerable amounts of formate (up to 23 mM) depending on the oxygen-stress conditions. We sequenced the 16S rRNA gene cloned from UR1 genomic DNA, compared it with that from 21 Bifidobacterium species and constructed a phylogenetic tree by alignment analysis. The most related species of strain UR1 was calculated to be Bifidobacterium animalis DSM 20104. DNA-DNA hybridizations between the genomes of strain UR1, B. animalis DSM 20104 and B. longum DSM 20219 showed only weak homology (27% and B. animalis . Therefore, we concluded that strain UR1 has to be regarded as a new species which we named Bifidobacterium lactis . This name honours the fact that the species has been isolated on several occasions from fermented milk.

Taxonomic Differentiation of Bacteriophages of Lactococcus lactis by Electron Microscopy, DNA-DNA Hybridization, and Protein Profiles

SUMMARY: Thirty-seven virulent and 19 temperate bacteriophages of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were classified in a taxonomic system on the basis of morphology, DNA-DNA hybridization, and protein composition. As judged from electron microscopy and susceptibility to cleavage by restriction endonucleases, the genome of all the bacteriophages investigated is composed of double stranded DNA. Seven virulent phage groups were recognized: types P034 (genome size 18·1 kilobase pairs, kb), P001 (20·2 kb), P008 (29·7 kb), P335 (36·4 kb), P026 (51·5 kb), P107 (51·5 kb), and P087 (54·5 kb). In addition, two temperate phage groups were established: types TP-40-3 (genome size 42·1 kb) and TP-936-1 (37·8 kb). Phages within each group revealed strong DNA homology and similar protein compositions, whereas no significant DNA homology and different proteins were found in phages of different groups. Virulent phages of group P335 exhibited strong DNA homology with the temperate phages of group TP-936-1.

Acetobacter europaeus sp. nov., a main component of industrial vinegar fermenters in central Europe

Summary A new species in the genus Acetobacter , for which we propose the name A. europaeus sp. nov., has been isolated and characterized in pure culture from high acid vinegar fermentations in Germany and Switzerland. In contrast to the other species of acetic acid bacteria, the isolated strains from industrial vinegar fermenters need acetic acid for growth. They could be cultivated on a special double layer agar with 4 to 8% acetic acid ( Entani et al., 1985). DNA-DNA hybridization between the DNA of 10 isolated A. europaeus strains as labelled DNA probe with the type strains of Acetobacter aceti, A. hansenii, A. liquefaciens, A. methanolicus, A. pasteurianus, A. xylinum, Gluconobacter oxydans and G. oxydans subsp. melanogenes indicated less than 25% DNA similarity. The important features of the new species are described. Acetobacter europaeus strain DES 11 (DSM 6160) is the type strain.

Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

Though the primary action of the cationic antibiotic polymyxin B is against the membrane of susceptible bacteria, severe morphological changes are detected in the cytoplasm. Using fluorescence microscopy and a mono-N-dansyl-polymyxin B derivative, we could demonstrate aggregations of the antibiotic with cellular material, possibly nucleic acids and/or ribosomes. These aggregations were only produced by minimum inhibitory or higher concentrations of the antibiotic as shown with Salmonella and Escherichia strains differing in their polymyxin susceptibility. The outer membrane of Salmonella typhimurium revealed characteristic blebs when treated with polymyxin B. This was investigated by the gentle methods of spray-freezing and freeze-etching. The obtained electron micrographs suggest that the polymyxin-induced blebs are projections of the outer monolayer of the outer membrane. A possible mechanism of penetration of polymyxin B through the cell envelope of gram-negative bacteria is presented. Images

Characterization of the d-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) from Bifidobacterium lactis

A D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase (Xfp) from the probiotic Bifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with D-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme. K(m) values for D-xylulose 5-phosphate and D-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (named xfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta and guaA, were localized adjacent to xfp on the B. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes in Mycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.

Simultaneous extraction and purification of a cell wall-associated peptidase and β-casein specific proteas fromStreptococcus cremoris AC1

SummaryA cell wall-associated, β-casein specific protease and a peptidase were purified simulta-neously fromStreptococcus cremoris AC1. The molecular weights are 145,000 daltons for the pro-tease and 36,000 for the peptidase. The protease has a pH optimum at 5.5-6 and a temperature optimum at 40° C. It is activated by 1 mM of Ca++ but severely inhibited by higher Ca++ concentrations. Further inhibitor studies indicate that the protease is probably a serin protease. The peptidase shows aminopeptidase activity and most effectively hydrolyses L-lysyl-p-nitroanilide, and to a lesser extent IMeucyl-, L-alanyl- and L-alanyl-L-alanyl-p-nitroanilide. No endopeptidase activity could be detected. The peptidase is irreversibly inhibited by EDTA.

Purification of an X-prolyl-dipeptidyl aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp. cremoris

SummaryThe β-casein specific cell wall proteolytic system of Lactococcus lactis subsp. cremoris P8-2-47 contains a metal-independent X-prolyl-dipeptidyl-aminopeptidase. Suitable substrates for its assay are Gly-Pro-nitroanilide and Ala-Pronitroanilide. It is suggested that the function of the enzyme is to cleave the proline-rich sequences of β-casein, as shown by the degradation of β-casomorphin. It is a serine proteinase with a monomer molecular mass of about 90 000 daltons, a temperature optimum of 45°–50°C, and a pH optimum of about 7.