Microarray based methods

Microarrays were developed following the availability of microbial whole genome sequences (Doolittle, 2002). Microarrays allow for the simultaneous analysis of every gene in the genome, either at the DNA or mRNA level (Ye et al., 2001).

Microarrays are constructed by depositing probes such as nucleic acids, proteins, carbohydrates, and antibodies on a solid surface such as glass. There are two types of commonly used DNA microarrays namely: PCR product - based DNA microarray and oligonucleotide-based DNA microarrays. PCR product-based microarrays are prepared by spotting the PCR amplicons onto a glass microscope slide. Although PCR based microarrays offer advantages such as ease of production and relative low cost, they are prone to cross-hybridisation between genes that are very similar (Wren

51 et al., 2002), which makes it difficult to detect genetic insertions, inversions, duplications or indeed point mutations (Dorrell et al., 2002). Unlike PCR arrays, oligonucleotide – based DNA arrays are generated by identifying areas of specificity within any gene sequence as targets for hybridisation. Knowledge of the sequence of the target DNA is required for synthesising complementary oligonucleotides. The advantages of oligonucleotide over PCR based microarrays include a reduction in cross-hybridisation and an increase in distinguishing homologous genes (Dorrell et al., 2002). To determine the presence of target genes, nucleic acid samples are labelled, either chemically or by an enzymatic reaction and then hybridized onto the array (Figure 1.13). Unbound and non-specifically bound samples are removed by washing using different stringency buffers. The remaining signal resulting from specific interactions between probes and target nucleic acids is measured. Only probes that hybridize to a labelled complementary target will yield a signal, thereby ascertaining the presence of the gene of interest in the sample (Huyghe et al., 2009).

52

Sample

A B

DNA/RNA DNA/RNA transformation

and labelling

Labelled nucleic acids mixed

Hybridisation onto microarray and scanning

Data analysis DNA/RNA

Figure 1.13 Microarray workflow

(http://grf.lshtm.ac.uk/microarrayoverview.htm)

Microarray technology has found wide applications in microbiology such as detection of differential gene expression, gene regulation as well as comparative genomics (Behr et al., 1999; Richmond et al., 1999; de Saizieu et al., 2000).

However, the limitation of microarray analyses is that they are restricted to the genes present in the reference strain(s) on the microarray, which renders rapid identification of acquired DNA in outbreak strains difficult. This thesis describes studies using oligonucleotide microarray to characterise serotypes in carriage and also estimate the prevalence of multiple carriage in Malawian adults and children.

Unlike Quellung or latex agglutination, microarray has the enhanced utility to detect

53 not only carriage of multiple strains of S. pneumoniae and their relative abundance but also detect the presence of antibiotic resistance genes, novel serotypes and other colonising microbial species in the nasopharynx (Newton et al., 2011, Turner et al., 2011). Microarray also offers the advantage of analysing large number of samples within a short period of time and can be optimised to detect non-viable organisms (Satzke et al., 2014).

1.7.2.2.1. S. pneumoniae capsular polysaccharide (SP-CPS) microarray The SP-CPS microarray is a novel molecular based serotyping technique developed by the bacterial microarray group at St George‟s (B G@S) in London. It was designed based on the availability of DNA sequences for all the genes responsible for the synthesis of the capsular polysaccharide (CPS), which determines serotypes.

The BμG@S SP-CPSmicroarray (http://www.bugs.sgul.ac.uk) contained several thousands of 60mer oligonucleotides probes, with 10 oligonucleotide probes per gene (Figure 1.14).

Oligonucleotide probes cps gene (+ve strand)

cps gene (-ve strand)

Figure 1.14 The CPSgene target regions of the 60-mer oligonucleotides

There are 10 oligonucleotides per CPS gene and these are spread along the length of the target CPS gene on both negative and positive strands and are randomly printed on the array. The use of multiple oligonucleotides per CPS gene plays an important role in minimising false positives due to cross hybridisation.

54 The long 60mer, in situ synthesised oligonucleotides DNA microarrays are known for their high sensitivity. They are also very tolerant of sequence mismatches and are thus suitable for the analysis of regions, which are highly polymorphic (Fenart et al., 2013). These probes detect a number of different entities (Hinds et al., 2009): (a) homology group identification (HGID) probes (Chapter 2, section 2.6.4.1) for determining serotype by the presence of capsular polysaccharide locus genes, with 10 oligonucleotide reporters per gene. There are 432 CPS genes known to date, however a particular serotype can only contain a subset of these genes (Newton et al., 2011); (b) serotype identification (STID) (Chapter 2, section 2.6.4.1) oligonucleotides designed to discriminate serotypes with identical HGID; (c) pathogen identification (PathID) probes (Chapter 2, section 2.6.4.1) for identifying bacterial species other than S. pneumoniae, that are commonly found in asymptomatic carriage; (d) antibiotic resistance (AbR) reporters (Chapter 2, section 2.6.4.1.1) for detecting the presence of antibiotic resistance genetic markers. The array used in this study was only designed to detect 10 antibiotic resistance genetic markers such as aphA3 (kanamycin resistance) (Werner et al., 2001), chloramphenicol acetyltransferase (cat) gene (chloramphenicol resistance), erythromycin methylase (ermB/C) genes (erythromycin resistance), macrolide efflux (mefA) gene (macrolide resistance) (Arpin et al., 1999, Ardanuy et al., 2005), streptothricin acetyltransferase (sat4) gene(streptothricin resistance) (Werner et al., 2001), and tetK/L/M/O genes as markers for tetracyclines and macrolides resistance (Marimon et al., 2006). Apart from detecting serotypes, the BμG@S SP-CPS microarray also offers enhanced utility for novel serotype discovery; nontypeable (NT) strains investigation, detection of multiple serotype carriage (Chapter 2, section 2.6.4.3) and performing comparative genomic hybridisation (arrayCGH) using

55 spTIGR4+R6 genome backbone (Hinds et al., 2009). The sensitivity of the fluorescent detection enables serotypes in the order of 1% relative abundance to be detected in multiple carriage samples. The discovery of novel serotypes is achieved by detecting HG profiles that do not match the CPSgene content of any of the currently known reference strain serotypes (Hinds et al., 2009).