The mechanisms responsible for macrolide resistance in Streptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14-and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A), erm(A), erm(B), erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids ( 63 KPWRQKGTGRAR 74 ) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.

Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14- and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low-copy vector, pACYC177, and expressed in several Escherichia coli K-12 strains. The location of the macrolide-resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane-associated pump proteins. The macrolide-resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E.coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA. Furthermore, intracellular accumulation of [14C]-erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin-sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer-specific synthesis using the polymerase chain reaction.

Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.

Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 g/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 g/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously ( Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 g/ml) lowered the ampicillin MIC to 3.67 g/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD ؉ -dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD The increasing incidence of drug resistance among bacterial pathogens, including Staphylococcus aureus, has stimulated the development of strategies targeting previously unexploited mechanisms of antibiotic action. Moreover, the emergence of vancomycin-resistant enterococci and drug-resistant Streptococcus pneumoniae has illustrated the necessity for antibacterials to combat multiply resistant gram-positive pathogens (19,20). Attractive targets for novel antimicrobial agents can be found among genes that are essential for bacterial survival. In an effort to identify genes essential for the growth of S. aureus, a collection of temperature-sensitive mutants has been generated (13). One of the mutant strains, NT64, was found to be complemented by genes encoding an NAD ϩ -dependent DNA ligase.DNA ligases are essential enzymes found in all bacteria that catalyze the formation of phosphodiester bonds at singlestrand breaks between adjacent 3Ј-OH and 5Ј-phosphate termini in double-stranded (ds) DNA (7,30). This activity plays an essential role in DNA replication, repair of damaged DNA, and recombination (11,15,17,18,26). Reports describing conditional lethal mutations in the ligase gene of Escherichia coli have confirmed the essentiality of this important enzyme, since mutants are deficient in both DNA replication and repair (1, 2).The DNA ligase family can be divided into two classes: those requiring ATP for adenylation (eukaryotic cells and phage), and those requiring NAD ϩ for adenylation, which include all known bacterial DNA ligases (7,18,21,23,25,26,29). Amino acid sequence comparisons indicate that NAD ϩ -dependent ligases are phylogenetically unrelated to the ATP-dependent DNA ligases. Eukaryotic, bacteriophage, and viral DNA ligases show little sequence homology to DNA ligases isolated from prokaryotes, with the exception of the conserved residues within the central cofactor-binding core (28,29). This suggests that bacterial DNA ligase may be a selective target for new antibacterials.The first step of DNA ligation in bacteria requires adenylation by the NAD ϩ cofactor of an ε-NH 2 group of lysine in the conserved KXDG motif at amino acids (aa) 112 to 115 (see