Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal D-alanyl-D-alanine of the nascent peptidoglycan with D-alanyl-D-lactate or D-alanyl-D-serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 g/ml and a MIC of vancomycin of 90 g/ml during vegetative growth. The vanH, vanA, and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in D-alanyl-D-lactate. No trace of precursors ending in D-alanyl-D-alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 g/ml), the MIC of teicoplanin remained in the range of 25 to 35 g/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 g/ml.Glycopeptide antibiotics are produced by actinomycetes and inhibit the synthesis of bacterial cell wall by blocking peptidoglycan assembly. They bind to the D-alanyl-D-alanine (DAla-D-Ala) C terminus of the nascent peptidoglycan and prevent it from being utilized in the following cross-linking reactions catalyzed by transglycosylases and transpeptidases (18,38,44). The structurally related glycopeptides vancomycin and teicoplanin have been used in clinical settings since 1958 and 1988, respectively. These drugs are still extensively used against multiresistant enterococci and methicillin-resistant staphylococci. Concern about vancomycin-resistant enterococci has been increasing during the last decade, and highly vancomycin resistant Staphylococcus aureus isolates have recently appeared in clinical specimens (10, 11).Two resistance phenotypes, VanA and VanB, have been extensively studied in enterococci and are considered of main importance, since the genes responsible for resistance (van genes) are inducible and transferable and confer high-level resistance to vancomycin (at concentrations up to 1,000 g/ml or more) (42). The vanA gene cluster also confers high-level resistance to teicoplanin (4,9,15,16) (16,45,51). The genes vanH, vanA/vanB, and vanX are organized in a cluster and are oft...

The internal transcribed spacers between the 16S and the 23S ribosomal RNA genes were used to discriminate species of the 16S rRNA group I of the genus Bacillus by PCR. The spacer-PCR fingerprints clearly discriminated the different species, except those closely related like the members of the 'B. cereus group' (B. cereus, B. thuringiensis and B. mycoides) and the species of the 'B. subtilis group' (B. amyloliquefaciens and B. licheniformis). Examining in more detail the shortest internal transcribed spacers, B. subtilis group species were distinguished by single-strand conformation polymorphism analysis, whereas B. mycoides was differentiated from B. cereus/B. thuringiensis by restriction analysis.

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains.