Stefan Schwarz

Nucleotide sequence and phylogeny of the tet(L) tetracycline resistance determinant encoded by plasmid pSTE1 from Staphylococcus hyicus.

The nucleotide sequence of the tetracycline resistance (tet) gene and its regulatory region, encoded by the plasmid pSTE1 from Staphylococcus hyicus, was determined. The tet gene was inducible by tetracycline and encoded a hydrophobic protein of 458 amino acids. Comparisons between the predicted amino acid sequences of the pSTE1-encoded Tet from S. hyicus and the previously sequenced Tet K variants from Staphylococcus aureus, Tet L variants from Bacillus cereus, Bacillus stearothermophilus, and Bacillus subtilis, Tet M variants from Streptococcus faecalis and Staphylococcus aureus as well as Tet O from Streptococcus mutans were performed. An alignment of Tet amino acid sequences revealed the presence of 30 conserved amino acids among these Tet variants. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated large evolutionary distances between the Tet M and Tet O variants on one hand and the Tet K and Tet L variants on the other hand. The pSTE1-encoded Tet proved to be closely related to the Tet L proteins originally found on small Bacillus plasmids. The observed extensive similarities in the nucleotide sequences of the tet genes and in the deduced Tet amino acid sequences allowed the assignment of the pSTE1-encoded Tet to the Tet proteins of class L.

Small antibiotic resistance plasmids in Staphylococcus intermedius

Antibiograms and plasmid profiles were evaluated for 116 Staphylococcus intermedius isolates collected from dogs in Germany and in the USA. Of the 26 S. intermedius isolates from Germany, 9 (34.6%) carried plasmids, while 20 (22.2%) of the 90 S. intermedius isolates from the USA were found to be plasmid-positive. Eight small resistance plasmids were identified and characterized using protoplast transformations and restriction endonuclease analyses. Five plasmids (3.8 and 3.9 kb) encoded for chloramphenicol resistance, 2 plasmids (each 2.5 kb) carried determinants for macrolide-lincosamide resistance, and one plasmid (4.5 kb) conferred resistance to tetracycline. Detailed restriction maps of these plasmids were constructed and served for structural comparisons with other small resistance plasmids found in staphylococci. These comparisons implied marked structural homologies with those prototype plasmids initially characterized in S. aureus of human origin.

Use of ribotyping, IS200 typing and plasmid analysis for the identification of Salmonella enterica subsp.enterica serovar typhimurium vaccine strain Zoosaloral H and its differentiation from wild type strains of the same serovar

Fifty Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates obtained from one vaccinated and three non-vaccinated poultry flocks as well as the commercially available vaccine strain Zoosaloral H and a S. Typhimurium reference strain were characterized genotypically to differentiate between S. Typhimurium live vaccine strain Zoosaloral H and wild type strains of the same serovar. Ribotyping revealed five different patterns one of which exclusively occurred in the vaccine strain. Seven different hybridization patterns could be observed by IS200-typing of the S. Typhimurium isolates; one of them was only detectable in the vaccine strain. Plasmid analysis showed that 51% of the S. Typhimurium isolates including the vaccine strain harboured large plasmids of approximately 60 MDa. Hybridization with a virulence gene probe identified only 48% of these large plasmids, including that of the vaccine strain, to carry this virulence-associated gene. However, restriction endonuclease analysis of the hybridizing plasmids showed that the virulence gene was located on HindIII fragments of different sizes in the plasmid of the S. Typhimurium vaccine strain Zoosaloral H and in the plasmids of the respective wild type S. Typhimurium isolates. Thus, ribotyping, IS200-typing and plasmid analysis represent at least three independent systems which allow the genotypic identification of the S. Typhimurium vaccine strain Zoosaloral H and its differentiation from wild type isolates of the same serovar.

Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius.

The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.

Pulsed-field gel electrophoretic identification of Salmonella enterica serovar Typhimurium live vaccine strain Zoosaloral H

Salmonella enterica subsp. enterica serovar Typhimurium (Salm. Typhimurium) live vaccine strain Zoosaloral H was characterized by pulsed‐field gel electrophoresis (PFGE). Each of the two suitable restriction enzymes, XbaI and SpeI, produced a unique restriction fragment pattern for this live vaccine strain which was not shared by field isolates of the same serovar. The characteristic fragment pattern proved to be stable during a 22 month observation period and was also not altered after animal passage of the vaccine strains. Thus PFGE analysis proved to be a helpful tool in the identification of Salm. Typhimurium live vaccine strain Zoosaloral H and its differentiation from wild‐type isolates of the same serovar.

Nucleotide sequence and phylogeny of a chloramphenicol acetyltransferase encoded by the plasmid pSCS7 from Staphylococcus aureus.

The nucleotide sequence of the chloramphenicol acetyltransferase gene (cat) and its regulatory region, encoded by the plasmid pSCS7 from Staphylococcus aureus, was determined. The structural cat gene encoded a protein of 209 amino acids, which represented one monomer of the enzyme chloramphenicol acetyltransferase (CAT). Comparisons between the amino acid sequences of the pSCS7-encoded CAT from S. aureus and the previously sequenced CAT variants from S. aureus, Staphylococcus intermedius, Staphylococcus haemolyticus, Bacillus pumilis, Clostridium difficile, Clostridium perfringens, Escherichia coli, Shigella flexneri, and Proteus mirabilis were performed. An alignment of CAT amino acid sequences demonstrated the presence of 34 conserved amino acids among all CAT variants. These conserved residues were considered for their possible roles in the structure and function of CAT. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated relatively large evolutionary distances between the CAT variants of enteric bacteria, Clostridium, Bacillus, and Staphylococcus species.

Antibiotic-resistance and plasmids in Staphylococcus hyicus isolated from pigs with exudative epidermitis and from healthy pigs☆

A total of 100 S. hyicus strains isolated from healthy piglets and piglets with exudative epidermitis originating from 100 different herds was examined for drug-resistance and prevalence of plasmids. Resistance to macrolide/linosamide antibiotics could be related to plasmids in 55 (93%) of the 59 resistant strains: A plasmid of 2.4 kb mediating resistance to macrolides and lincosamides was observed in 25 strains, and a plasmid of 11.5 kb mediating resistance to both macrolides/lincosamides and tetracycline was observed in 30 strains. A plasmid with a molecular weight of 4.5 kb was shown by curing experiments to be associated with resistance to tetracycline in 12 strains. All together, 47 strains were resistant to tetracycline. In 42 (89%) of these strains tetracycline-resistance was found to be encoded by plasmids. Fifty six strains were resistant to streptomycin, and resistance was associated with the presence of a 4.4 kb plasmid in 17 strains studied. Resistance to penicillin, observed in 44 strains, and resistance to kanamycin, observed in 15 strains, could not be related to plasmids in any of these strains. The 11.5 kb plasmid was observed in 39% of the strains isolated from piglets with EE, and in 7% of the strains isolated from healthy piglets. Despite its higher prevalence in strains from piglets with EE, the 11.5 kb plasmid could not be shown to encode production of capsule or exfoliative substances: factors which might play a role in the development of exudative epidermitis in piglets.

Molecular cloning, purification, and properties of a plasmid-encoded chloramphenicol acetyltransferase from Staphylococcus haemolyticus.

A small chloramphenicol resistance (Cmr) plasmid of approximately 3.75 kb, designated pSCS5, was isolated from Staphylococcus haemolyticus. This plasmid encoded an inducible chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). The cat gene of pSCS5 was cloned into the Escherichia coli plasmid vector pBluescript SKII+. It differed in its nucleotide sequence and deduced amino acid sequence from the cat genes described previously in staphylococci and other gram-positive bacteria. The CAT enzyme was purified from cell-free lysates by ammonium sulfate precipitation, ion-exchange chromatography, and fast protein liquid chromatography. The native enzyme had an Mr of 70,000 and was composed of three identical subunits, each with an Mr of approximately 23,000. Its isoelectric point was at pH 6.15. CAT from pSCS5 exhibited Km values of 2.81 and 51.8 microM for chloramphenicol and acetyl coenzyme A, respectively. The optimum pH for activity was 7.8. CAT encoded by pSCS5 proved to be relatively heat stable, but sensitive to mercury ions. The observed differences in the nucleotide sequence and the biochemical characteristics of the enzyme allowed the identification of the pSCS5-encoded CAT from S. haemolyticus as a CAT variant different from those described previously in gram-positive bacteria. Images

Structure and putative origin of a plasmid from Staphylococcus hyicus that mediates chloramphenicol and streptomycin resistance

The structure and functional organization of Staphylococcus hyicus plasmid pSCGp3EB that mediates chloramphenicol and streptomycin resistance (CmrSmr) is described and compared with another CmrSmr plasmid, pSCS12, from Staphylococcus sciuri. Both plasmids appeared to be formed by co‐integrate formation between plasmids that very closely resemble the chloramphenicol resistance (Cmr) plasmid pC221 and the streptomycin resistance (Smr) plasmid pS194. In addition to the established recombination site B (RSB) in pC221 and pS194, another area suitable for recombination immediately downstream of the cat gene in pC221 and upstream of the str gene in pS194 has been identified. Co‐integration at these sites would lead to the structures we have observed in the wild‐type CmrSmr plasmids pSCGp3EB and pSCS12.

Plasmid‐encoded resistance to tetracycline in Staphylococcus haemolyticus

A small 4.35 kb plasmid, designated pSTS4, was isolated from a multiresistant Staphylococcus haemolyticus culture. It conferred resistance to tetracycline as shown by protoplast transformation. pSTS4 was further characterized by restriction endo‐nuclease analyses and a preliminary restriction map constructed. It shared some structural similarities with previously described small TcR plasmids from other staphylococcal species of human and animal origin. pSTS4 encoded two proteins of approximately 37 kDa and 52 kDa as revealed by combined in vitro transcription/translation assays.