Observation of Pneumococcal Phase Variation in Colony Morphology

Background

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children worldwide (Walker et al., 2013). The success of this pathogen in its adaptation to various ecological niches of human host depends on its remarkable phenotypic plasticity (Croucher et al., 2013; Johnston et al., 2014a), which has been reflected by the inter-strain antigenic variation in the capsular polysaccharides and surface proteins (Croucher et al., 2013 and 2011), acquisition of new virulence factors (Park et al., 2012), extensive drug resistance (Croucher et al., 2014) within the species. Natural genetic transformation is a well-known mechanism contributing to this phenotypic plasticity (Johnston et al., 2014b). Moreover, S. pneumoniae is also capable of spontaneous phase variation between opaque and transparent colony phenotypes, a widespread phenomenon in microbial pathogens (van der Woude, 2011). The opaque variants, which have more capsule and less teichoic acid, are more virulent in the lung and bloodstream; the transparent counterparts, which express less capsule and have more teichoic acid, are more adaptive to the nasopharynx (Kim and Weiser, 1998; Weiser et al., 1994; Manso et al., 2014; Li et al., 2016). Our recent studies have revealed that pneumococcal phase variation between two colony phenotypes is determined by DNA inversion between the methyltransferase hsdS genes in the colony opacity determinant (cod) locus (Feng et al., 2014; Li et al., 2016).

The protocol we present here describes the complete experimental procedure from preparing a bacterial stock to obtaining an image of colony morphology as described in our recent study (Li et al., 2016). Animal blood is commonly added to agar medium to promote growth of S. pneumoniae, but the color of the blood makes it difficult to differentiate opaque and transparent colonies by microscopic approach. Instead, catalase is supplemented to agar plates to culture S. pneumoniae by neutralizing the inhibition effect of hydrogen peroxide (produced by the pneumococci themselves) on pneumococcal growth when it comes to observe and document colony morphologies by dissection microscope (Kim and Weiser, 1998; Weiser et al., 1994; Manso et al., 2014; Li et al., 2016). Since colony morphology phenotypes can be indicative of bacterial physiological and pathogenic properties, this protocol may offer a valuable method to study the impact of genetic and epigenetic elements or environmental conditions on bacterial biology and disease pathogenesis.