The discovery and development of the β-lactam antibiotics are among the most powerful and successful achievements of modern science and technology. Since Fleming's accidental discovery of the penicillin-producing mold, seventy years of steady progress has followed, and today the β-lactam group of compounds are the most successful example of natural product application and chemotherapy. Following on the heels of penicillin production by Penicillium chrysogenum came the discoveries of cephalosporin formation by Cephalosporium acremonium, cephamycin, clavam and carbapenem production by actinomycetes, and monocyclic β-lactam production by actinomycetes and unicellular bacteria. Each one of these groups has yielded medically-useful products and has contributed to the reduction of pain and suffering of people throughout the world. Research on the microbiology, biochemistry, genetics and chemistry of these compounds have continued up to the present with major contributions being made by both individual and collaborative groups from industry and academia. The discovery of penicillin not only led to the era of the wonder drugs but provided the most important antibiotics available to medicine. Continued efforts have resulted in the improvement of these compounds with respect to potency, breadth of spectrum, activity against resistant pathogens, stability and pharmacokinetic properties. On the research front, major advances are being made on structural and regulatory biosynthetic genes and metabolic engineering of the pathways involved. New semisynthetic compounds especially those designed to combat resistance development are being examined in the clinic, and unusual non-antibiotic activities of these compounds are being pursued. Although seventy years of age, the β-lactams are not yet ready for retirement.

The beta-lactam antibiotics: past, present, and future.

Lincosamide antibiotics include lincomycin, naturally produced by several actinomycetes, and clindamycin, a semisynthetic derivative obtained by the chlorination of lincomycin. These antibiotics are active against many gram-positive cocci and anaerobes; they inhibit protein synthesis by blocking the peptidyltransferase activity of the 50S subunit of the bacterial ribosome (11). Resistance to lincosamides is usually due to alteration of the ribosome following the N6 dimethylation of a specific adenine in the 23S rRNA, which confers cross-resistance to macrolide, lincosamide, and streptogramin B type antibiotics, i.e., the MLSB phenotype (22, 32). In contrast to this broad-spectrum resistance, resistance specific to lincosamides, gained by bacterial modification of those antibiotics, has been reported. Phosphorylation (1) and nucleotidylation (2, 26) of the hydroxyl group in position 3 of lincosamide molecules (24, 26) have been detected in several species of Streptomyces. Inactivation of lincosamides was also observed in strains of staphylococci, streptococci, enterococci, and lactobacilli of animal origin (10, 12, 13) and in staphylococci isolated from humans (5, 20, 21). Clinical isolates of Staphylococcus haemolyticus BM4610 and Staphylococcus aureus BM4611 are highly resistant to lincomycin (MIC = 64 μg/ml) and are apparently susceptible to clindamycin (MIC = 0.12 μg/ml). In these strains, lincosamide O-nucleotidyltransferases encoded by two closely related genes named linA (lincosamide inactivation nucleotidylation) and linA′, respectively, were characterized (4, 5). These genes encode two 161-amino-acid isoenzymes that differ by 14 amino acids. These enzymes inactivate lincomycin and clindamycin by converting them to lincomycin 3-(5′-adenylate) and clindamycin 4-(5′-adenylate) by using ATP, GTP, CTP, or UTP as a nucleotidyl donor and MgCl2 as a cofactor (5). The distribution of linA and linA′ genes was studied by using DNA-DNA hybridization, and related sequences were found in strains belonging to various species of staphylococci (20).

In this paper, we report the nucleotide sequence of a new linB gene that confers resistance to lincosamides on a clinical strain of Enterococcus faecium, HM1025, by inactivating the compounds, and we further report on our study of the biochemical mechanism of the resistance.

A New Resistance Gene, linB, Conferring Resistance to Lincosamides by Nucleotidylation in Enterococcus faecium HM1025

Comparative aspects of development and differentiation in actinomycetes.

Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 microg/ml) but susceptible to clindamycin (MIC = 0.12 microg/ml) and erythromycin (MIC = 0.06 microg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuA(N2), LnuA, and LnuA' lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.

New lnu(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in Streptococcus agalactiae UCN36

In den letzten Jahren haben sich Chemie und Biochemie der Makrolide eindrucksvoll weiterentwickelt. In dieser Ubersicht ist versucht worden, die wesentlichen Arbeiten auf den Gebieten der Strukturermittlung, der chemischen Synthese, der Biochemie und der antimikrobiellen Aktivitat vor allem der Polyoxomakrolid-Antibiotica zusammenzufassen. Im Vordergrund stehen dabei die Fortschritte der Makrolid-Synthese.

Makrolide. Neuere Fortschritte ihrer Chemie und Biochemie

Streptomyces sp. strain No. K-245 was found to transform maridomycin III into four derivatives (A1, A2, A3 and A4) in addition to the transformation products reported previously. Isolation of the main product A1 was carried out by column chromatography on silica gel developed with CHCl3-MeOH (19:1). From the partial investigation of the structure of A1 it proved to have a C 18-aldehyde group and C 4"-propionyl group but no antimicrobial activity. The relationships between A group's derivatives and known derivatives of maridomycin III are also discussed.

Microbial transformation of antibiotics

Three transformation products of maridomycin (MDM) III, a macrolide antibiotic, by Streptomyces lavendulae were isolated by silica gel and alumina chromatography, and designated as spots 1 (starting MDM III), 2, 3 and 4, in the order of their decreasing Rf values on thin-layer chromatogram. By mass- and NMR-spectrometry and thin-layer chromatography, spot 2 was identified as 18-dihydro-MDM III, spot 3 as 4''-depropionyl-MDM III, and spot 4 as 18-dihydro-4''-depropionyl-MDM III. The relationship between starting MDM III and these transformation products were also discussed.

I. Isolation and characterization of the transformation products of maridomycin III.

Microbial transformation of antibiotics. III. Reacylation of 4"-depropionyl maridomycin III into maridomycin V (maridomycin K) by Streptomyces sp. strain no. K-342.

Shinya Mori

Papers

Microbial transformation of antibiotics

I. Isolation and characterization of the transformation products of maridomycin III.

Microbial transformation of antibiotics. III. Reacylation of 4"-depropionyl maridomycin III into maridomycin V (maridomycin K) by Streptomyces sp. strain no. K-342.