Interaction of Streptococcus pneumoniae and Moraxella catarrhalis: investigation of the indirect pathogenic role of beta-lactamase-producing moraxellae by use of a continuous-culture biofilm system

Abstract

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.

FIG. 1

Biofilm and effluent titers of S. pneumoniae (• and ○, respectively) and M. catarrhalis (▴ and ▵, respectively) (β-lactamase negative [a, c, and e] and β-lactamase positive [BRO1] [b, d, and f]) grown together on Sorbarod biofilms in the presence of benzylpenicillin (a and b), amoxicillin (c and d), and amoxicillin-clavulanic acid (e and f). Individual filters were inoculated with mixtures of the two organisms and, after 24 h, were exposed to a single concentration of the antibiotic for 18 h. Effluent was collected for 15 min, the biofilm was sacrificed, and the titers of pneumococci and moraxellae were determined on selective agar. Titers in biofilm are expressed as total recoverable CFU per biofilm. Titers in effluent are expressed as CFU per milliliter.

FIG. 2

β-Lactamase activities in the supernatant fractions of biofilm (■) and effluent (⧫) preparations (Fig. 1) with the mixtures of the pneumococcus and strain BRO1. The supernatant was obtained by centrifuging both biofilms after vortex mixing and effluent at 3,500 rpm for 10 min. β-Lactamase activity (expressed as nanomoles of nitrocefin hydrolyzed per minute) was determined as described in the text. (a, benzylpenicillin; b, amoxicillin; c, amoxicillin-clavulanic acid).

FIG. 3

(a) Titers of strain BRO1 grown in broth culture and biofilm (plus biofilm effluent) collected at various times after inoculation. Titers in biofilm are expressed as recoverable CFU per biofilm; titers in effluent are expressed as CFU per milliliter. (b) β-Lactamase activity (nanomoles of nitrocefin hydrolyzed per minute) in the supernatants of the same specimens. ■, broth; ▴, biofilm; ▵, effluent.