Effects of cigarette smoke condensate on clarithromycin-mediated inhibition of biofilm formation and related alterations in resistance gene expression by Streptococcus pneumonia

Abstract

Streptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium that frequently colonises the nasopharynx of healthy humans. In susceptible hosts, especially children under 5 years with underdeveloped immune systems and the elderly, this asymptomatic colonisation can lead to development of severe disease such as pneumonia and meningitis. In many cases, disease severity is linked to the inability of the infection to be successfully treated, possibly due to formation of bacterial biofilms. In this context, cigarette smoking, which is a well-recognised risk factor for development of severe pneumococcal disease, also promotes biofilm formation by various types of bacterial pathogens. Notwithstanding poor penetration of biofilms by antibiotics, bacteria within biofilms are exposed to low levels of antibiotics, which promote gene modifications that mediate antibiotic resistance. However, little is known about the effects of exposure of the pneumococcus to cigarette smoke on the induction of pre-existing antibiotic resistance genes, specifically those that mediate resistance to macrolide antibiotics. In addition to measuring bacterial growth and biofilm formation, the research described in this dissertation was designed primarily to investigate the effects of exposure of different strains of the pneumococcus to cigarette smoke condensate (CSC) on the expression of genes which mediate resistance to macrolide antibiotics, specifically the erm(B) and mef(A) genes. The bacterial strains used were 172 (macrolide-susceptible), 521 [macrolide-resistant, mef(A) efflux pump-expressing] and 2507 [macrolide-resistant, erm(B) ribosomal methylase-expressing], all belonging to serotype 23F. In addition, the effects of exposure of all three strains of the pneumococcus to CSC on the expression of the SP2003 gene were also investigated. This gene encodes an ABC-type transporter, expression of which has been linked to antibiotic resistance. All three strains of the pneumococcus were exposed to CSC (80 and 160 μg/mL) and sub-minimal concentrations of clarithromycin individually or in combination, followed by measurement of growth, biofilm formation and gene expression. Bacterial growth was measured using spectrophotometric and colony counting procedures, biofilm formation by a crystal violet-based spectrophotometric method, and gene expression [(mef(A), erm(B) and SP2003)] using real-time qPCR. Exposure of all three strains of the pneumococcus to either CSC, at both concentrations used, or to clarithromycin alone, resulted in a transient inhibition of growth which persisted for several hours and was followed by a rebound. Exposure to combinations of the antibiotic and CSC resulted in prolongation of the lag phase, particularly in the case of strain 172. Augmentation of biofilm formation was observed following exposure of all three strains of the pneumococcus to CSC, while exposure of strain 172 to clarithromycin inhibited biofilm formation, which was partly attenuated by CSC. In the case of gene expression, exposure to clarithromycin alone caused significant upregulation of expression of the macrolide-resistance genes, mef(A) and erm(B), by strains 521 and 2507 respectively, as expected. Exposure of strain 2507 to the combination of clarithromycin and CSC resulted in significant augmentation of expression of the erm(B) gene relative to the expression level noted with clarithromycin alone. This augmentative effect of CSC on gene expression was not, however, evident in the case of the mef(A) gene. In addition, and somewhat surprisingly, exposure of strain 2507 to CSC only at 160 μg/mL resulted in a significant increase in erm(B) gene expression. In the case of the SP2003 gene, exposure of all three strains of the pneumococcus to CSC resulted in significant upregulation of this gene, probably as a stress response linked to elimination of smoke-derived toxicants, while exposure to clarithromycin alone resulted in modest upregulation, compatible with a role for SP2003 in mediating macrolide resistance. In conclusion, the pathogen-targeted effects of CSC described in this dissertation provide additional insights into the mechanisms by which cigarette smoking impacts negatively on the outcome of pneumococcal infections by undermining the therapeutic efficacy of macrolide antibiotics.