Trimethoprim resistance in Streptococcus pneumoniae can be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC 50 ], 4.2 M) than was the DHFR from strain CP1015 (IC 50 , 0.09 M). However, K m values indicated a lower affinity for the enzyme's natural substrates (K m for dihydrofolate [DHF], 3.1 M for CP1015 and 27.5 M for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, K m values for DHF were reduced (9.2 to 15.2 M), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC 50 of up to 13.7 M). It is postulated that carriage of the mutation M53-Iwhich, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR-is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates.

Azithromycin is an azalide antibiotic with excellent in vitro activity against a wide variety of oral bacteria. It has a long half-life, good tissue penetration and is preferentially taken up by phagocytes. We investigated the microbiological efficacy of azithromycin as an adjunct to the non-surgical treatment of adult chronic periodontitis; its clinical efficacy is dealt with in a separate paper. 46 patients were treated in a double-blind placebo controlled trial. Microbiological assessment of the same periodontal pocket (initially > 6 mm) was made at weeks 0, 2, 3, 6, 10 and 22. Either azithromycin 500 mg 1 x daily for 3 days or placebo was given at week 2. Particular attention was paid to the numbers of black pigmented anaerobes and spirochaetes present since these are the most commonly implicated pathogens in periodontal disease. Pigmented anaerobes were significantly reduced at weeks 3 and 6 in patients who received azithromycin compared to placebo and remained lower, although not significantly so, throughout the study. Counts of spirochaetes were significantly reduced throughout the study in patients who received azithromycin compared to placebo. Our microbiological study suggests that azithromycin may be useful as an adjunct in the treatment of periodontal disease.

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Sulfonamide resistance in

The in-vitro activities of azithromycin and erythromycin were compared against 689 clinical isolates, including Gram-positive cocci, Haemophilus influenzae, and anaerobes. Of the 100 methicillin-susceptible isolates of Staphylococcus aureus tested, 77% were susceptible to 1 mg/l azithromycin and 0.5 mg/l erythromycin, whereas 22% were resistant to 32 mg/l of both compounds. All methicillin-resistant S. aureus isolates were highly resistant to both macrolides (MIC greater than 64 mg/l). Coagulase-negative staphylococci showed a wide range of susceptibilities to both compounds; MIC50 values for azithromycin and erythromycin for all isolates were 0.5 and 0.25 mg/l, respectively. With the exception of enterococci, both macrolides showed similar activity against streptococci; MIC90 values for both group A and group B streptococci were 0.03 and 0.06 mg/l for erythromycin and azithromycin, respectively. Azithromycin was less active than erythromycin against enterococci, with mode MICs of 4.0 and 1.0 mg/l, respectively; about 20% of isolates were highly resistant to both compounds. Azithromycin was substantially more active than erythromycin against H. influenzae; 41% of isolates were inhibited by 0.5 mg/l azithromycin and all isolates were inhibited by 2 mg/l. The MIC90 for erythromycin was 8 mg/l; 36% of isolates required concentrations of greater than or equal to 4 mg/l for inhibition. The anaerobic bacteria tested showed similar susceptibility to both azithromycin and erythromycin.