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Capsule phase variants were isolated from serotype 8 and serotype 37 pneumococcal sorbarods. Sequence duplications within the essential capsule genes - cap8E (type 8) and tts (type 37) - were found to introduce frameshifts and generate acapsular phenotypes. Capsular revertants possessed wild-type cap8E and tts genes, indicating the precise excision of these duplications. Reversion frequencies (OFF-ON) fit a linear relationship between log(frequency of reversion) and log(length of duplication), previously found for serotype three pneumococci [Waite, R. D., Struthers, J. K. & Dowson, C. G. (2001). Mol Microbiol 42, 1223-1232]. This study provides evidence that capsule phase variation can occur in pneumococcal serotypes with either simple (one to three genes) or complex capsule-encoding loci (12 genes). Given the key role of CapE (the first monosaccharide transferase) in other clinically important pneumococci, such as serotypes 14 and 19F with complex capsular loci, the observed duplication within cap8E suggests that capsule phase variation could be controlled by tandem sequence duplication in capE homologues in other pneumococcal serotypes that construct their capsules through polymerization of lipid-linked intermediates.

The molecular genetic basis of high-frequency serotype 3 capsule phase variation in Streptococcus pneumoniae (the pneumococcus) was investigated. Pneumococci were grown in sorbarod biofilms at 34 degrees C to mimic nasopharyngeal carriage. Different type 3 pneumococci commonly associated with invasive disease generated apparently random tandem duplications of 11-239 bp segments within the cap3A gene of the type 3 capsule locus. These duplications alone were found to be responsible for high-frequency capsule phase variation, in which (phase off) acapsular variants possessed duplications within cap3A, and (phase on) capsular revertants possessed wild-type cap3A genes, indicating the precise excision of the duplication. Additionally, the frequency of phase reversion (off to on) was found to exhibit a linear relationship between (log) frequency of reversion and (log) length of duplication. This apparently random duplication giving rise to phase variation is in stark contrast to the 'preprogrammed' contingency genes in many Gram-negative organisms that possess homopolymeric sequence repeats or motifs for site-specific recombination.

The majority of clinical isolates of Moraxella catarrhalis produce beta-lactamase. The role of this enzyme in the phenomenon of indirect pathogenicity, in which a true pathogen such as Streptococcus pneumoniae is protected from the action of certain beta-lactam antibiotics, is well recognized. By using a simple continuous-culture biofilm system, it has been shown that the pneumococcus attains high titers in excess of 10(12) CFU/biofilm; furthermore, the penicillin-sensitive pneumococcus used remained susceptible to a range of beta-lactam antibiotics in these biofilms (R. K. Budhani and J. K. Struthers, J. Antimicrob. Chemother. 40:601-602, 1997). This system was used to characterize the antibiotic susceptibility of this isolate when grown with beta-lactamase-negative or -positive moraxellae. When grown with beta-lactamase-producing moraxellae in the presence of either benzylpenicillin or amoxicillin, the pneumococcus was protected in the range of the antibiotic concentrations to which it would be considered resistant. With amoxicillin-clavulanic acid the titers of the two organisms collapsed at the antibiotic concentration at which moraxellae became susceptible. The levels of beta-lactamase activity in cell-free supernatants of broth culture, in biofilm, and in biofilm effluent revealed distinct differences in this activity; levels in biofilm were significantly lower than those in broth culture supernatants. The system appears suitable for studying organisms under antibiotic stress and for investigating the interactions of bacteria under such conditions.